Staphylococcus aureus genes and polypeptides

ABSTRACT

The present invention relates to 11 novel genes from  S. aureus  and the polypeptides they encode. Also provided as are vectors, host cells, antibodies and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of  S. aureus  polypeptide activity. The invention additionally relates to diagnostic methods for detecting  Staphylococcus  nucleic acids, polypeptides and antibodies in a biological sample. The present invention further relates to novel vaccines for the prevention or attenuation of infection by  Staphylococcus.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of and claims priority under 35 U.S.C.§ 120 to U.S. application Ser. No. 09/830,217, filed Jan. 15, 2002 nowU.S. Pat. No. 6,521,441, which is the National Stage of InternationalApplication No. PCT/US99/06199, filed Mar. 18, 1999, which claimspriority to U.S. Provisional Application Nos. 60/084,674, filed May 7,1998, 60/080,296, filed Apr. 1, 1998, and 60/078,682, filed Mar. 20,1998. All of the foregoing applications are hereby incorporated byreference herein.

FIELD OF THE INVENTION

The present invention relates to novel Staphylococcus aureus genes (S.aureus) nucleic acids and polypeptides. Also provided are vectors, hostcells and recombinant methods for producing the same. Further providedare diagnostic methods for detecting Staphylococcus aureus using probes,primers, and antibodies to the S. aureus nucleic acids and polypeptidesof the present invention. The invention further relates to screeningmethods for identifying agonists and antagonists of S. aureuspolypeptide activity and to vaccines using S. aureus nucleic acids andpolypeptides.

BACKGROUND OF THE INVENTION

The genus Staphylococcus includes at least 20 distinct species. (For areview see Novick, R. P., The Staphylococcus as a Molecular GeneticSystem in MOLECULAR BIOLOGY OF THE STAPHYLOCOCCI, 1-37 (R. Novick, Ed.,VCH Publishers, New York (1990)). Species differ from one another by 80%or more, by hybridization kinetics, whereas strains within a species areat least 90% identical by the same measure.

The species S. aureus, a gram-positive, facultatively aerobic,clump-forming cocci, is among the most important etiological agents ofbacterial infection in humans, as discussed briefly below.

Human Health and S. aureus

Staphylococcus aureus is a ubiquitous pathogen. See, e.g., Mims et al.,MEDICAL MICROBIOLOGY (Mosby-Year Book Europe Limited, London, UK 1993).It is an etiological agent of a variety of conditions, ranging inseverity from mild to fatal. A few of the more common conditions causedby S. aureus infection are burns, cellulitis, eyelid infections, foodpoisoning, joint infections, neonatal conjunctivitis, osteomyelitis,skin infections, surgical wound infection, scalded skin syndrome andtoxic shock syndrome, some of which are described further below.

Burns: Burn wounds generally are sterile initially. However, theygenerally compromise physical and immune barriers to infection, causeloss of fluid and electrolytes and result in local or generalphysiological dysfunction. After cooling, contact with viable bacteriaresults in mixed colonization at the injury site. Infection may berestricted to the non-viable debris on the burn surface (“eschar”), itmay progress into full skin infection and invade viable tissue below theeschar and it may reach below the skin, enter the lymphatic and bloodcirculation and develop into septicemia. S. aureus is among the mostimportant pathogens typically found in burn wound infections. It candestroy granulation tissue and produce severe septicemia.

Cellulitis: Cellulitis, an acute infection of the skin that expands froma typically superficial origin to spread below the cutaneous layer, mostcommonly is caused by S. aureus in conjunction with S. pyrogenes.Cellulitis can lead to systemic infection. In fact, cellulitis can beone aspect of synergistic bacterial gangrene. This condition typicallyis caused by a mixture of S. aureus and microaerophilic streptococci. Itcauses necrosis and treatment is limited to excision of the necrotictissue. The condition often is fatal.

Eyelid infections: S. aureus is the cause of styes and of sticky eye” inneonates, among other eye infections. Typically such infections arelimited to the surface of the eye, and may occasionally penetrate thesurface with more severe consequences.

Food poisoning: Some strains of S. aureus produce one or more of fiveserologically distinct, heat and acid stable enterotoxins that are notdestroyed by digestive process of the stomach and small intestine(enterotoxins A-E). Ingestion of the toxin, in sufficient quantities,typically results in severe vomiting, but not diarrhea. The effect doesnot require viable bacteria. Although the toxins are known, theirmechanism of action is not understood.

Joint infections: S. aureus infects bone joints causing diseases suchosteomyelitis. See, e.g., R. Cunningham et al., (1996) J. Med.Microbiol. 44:157-164.

Osteomyelitis: S. aureus is the most common causative agent ofhaematogenous osteomyelitis. The disease tends to occur in children andadolescents more than adults and it is associated with non-penetratinginjuries to bones. Infection typically occurs in the long end of growingbone, hence its occurrence in physically immature populations. Mostoften, infection is localized in the vicinity of sprouting capillaryloops adjacent to epiphysis growth plates in the end of long, growingbones.

Skin infections: S. aureus is the most common pathogen of such minorskin infections as abscesses and boils. Such infections often areresolved by normal host response mechanisms, but they also can developinto severe internal infections. Recurrent infections of the nasalpassages plague nasal carriers of S. aureus.

Surgical Wound Infections: Surgical wounds often penetrate far into thebody. Infection of such wound thus poses a grave risk to the patient. S.aureus is the most important causative agent of infections in surgicalwounds. S. aureus is unusually adept at invading surgical wounds;sutured wounds can be infected by far fewer S. aureus cells then arenecessary to cause infection in normal skin. Invasion of surgical woundcan lead to severe S. aureus septicemia. Invasion of the blood stream byS. aureus can lead to seeding and infection of internal organs,particularly heart valves and bone, causing systemic diseases, such asendocarditis and osteomyelitis.

Scalded Skin Syndrome: S. aureus is responsible for “scalded skinsyndrome” (also called toxic epidermal necrosis, Ritter's disease andLyell's disease). This diseases occurs in older children, typically inoutbreaks caused by flowering of S. aureus strains produce exfoliation(also called scalded skin syndrome toxin). Although the bacteriainitially may infect only a minor lesion, the toxin destroysintercellular connections, spreads epidermal layers and allows theinfection to penetrate the outer layer of the skin, producing thedesquamation that typifies the diseases. Shedding of the outer layer ofskin generally reveals normal skin below, but fluid lost in the processcan produce severe injury in young children if it is not treatedproperly.

Toxic Shock Syndrome: Toxic shock syndrome is caused by strains of S.aureus that produce the so-called toxic shock syndrome toxin. Thedisease can be caused by S. aureus infection at any site, but it is toooften erroneously viewed exclusively as a disease solely of women whouse tampons. The disease involves toxemia and septicemia, and can befatal.

Nocosomial Infections: In the 1984 National Nocosomial InfectionSurveillance Study (“NNIS”) S. aureus was the most prevalent agent ofsurgical wound infections in many hospital services, including medicine,surgery, obstetrics, pediatrics and newborns.

Other Infections: Other types of infections, risk factors, etc.involving S. aureus are discussed in: A. Trilla (1995) J. Chemotherapy3:37-43; F. Espersen (1995) J. Chemotherapy 3:11-17; D. E. Craven (1995)J. Chemotherapy 3:19-28; J. D. Breen et al. (1995) Infect. Dis. Clin.North Am. 9(1):11-24 (each incorporated herein in their entireties).

Resistance to Drugs of S. aureus Strains

Prior to the introduction of penicillin the prognosis for patientsseriously infected with S. aureus was unfavorable. Following theintroduction of penicillin in the early 1940s even the worst S. aureusinfections generally could be treated successfully. The emergence ofpenicillin-resistant strains of S. aureus did not take long, however.Most strains of S. aureus encountered in hospital infections today donot respond to penicillin; although, fortunately, this is not the casefor S. aureus encountered in community infections.

It is well known now that penicillin-resistant strains of S. aureusproduce a lactamase which converts penicillin to pencillinoic acid, andthereby destroys antibiotic activity. Furthermore, the lactamase geneoften is propagated episomally, typically on a plasmid, and often isonly one of several genes on an episomal element that, together, confermultidrug resistance.

Methicillins, introduced in the 1960s, largely overcame the problem ofpenicillin resistance in S. aureus. These compounds conserve theportions of penicillin responsible for antibiotic activity and modify oralter other portions that make penicillin a good substrate forinactivating lactamases. However, methicillin resistance has emerged inS. aureus, along with resistance to many other antibiotics effectiveagainst this organism, including aminoglycosides, tetracycline,chloramphenicol, macrolides and lincosamides. In fact,methicillin-resistant strains of S. aureus generally are multiply drugresistant.

Methicillian-resistant S. aureus (MRSA) has become one of the mostimportant nosocomial pathogens worldwide and poses serious infectioncontrol problems. Today, many strains are multiresistant againstvirtually all antibiotics with the exception of vancomycin-typeglycopeptide antibiotics.

Recent reports that transfer of vancomycin resistance genes fromenterococci to S. aureus has been observed in the laboratory sustainthat fear that MRSA might become resistant against vancomycin, too, asituation generally considered to result in a public health disaster.MRSA owe their resistance against virtually all β-lactam antibiotics tothe expression of an extra penicillin binding protein (PBP) 2a, encodedby the mecA gene. This additional very low affinity pbp, which is foundexclusively in resistant strains, appears to be the only pbp stillfunctioning in cell wall peptidoglycan synthesis at β-lactamconcentrations high enough to saturate the normal set of S. aureus pbp1-4. In 1983 it was shown by insertion mutagenesis using transposonTn551 that several additional genes independent of mecA are needed tosustain the high level of methicillin resistance of MRSA. Interruptionof these genes did not influence the resistance level by interferingwith PBP2a expression, and were therefore called fem (factor essentialfor expression of methicillin resistance) or aux (auxiliary genes).

In the meantime six fem genes (femA- through F) have been described andthe minimal number of additional aux genes has been estimated to be morethan 10. Interference with fema and femB results in a strong reductionof methicillin resistance, back to sensitivity of strains without PBP2a.The fem genes are involved in specific steps of cell wall synthesis.Consequently, inactivation of fem factors induce β-lactamhypersensitivity in already sensitive strains. Both femA and femB havebeen shown to be involved in peptidoglycan pentaglycine interpeptidebridge formation. FemA is responsible for the formation of glycines 2and 3, and femB is responsible for formation of glycines 4 and 5. S.aureus may be involved in the formation of a monoglycine muropeptideprecursors. FemC-F influence amidation of the iso-D-glutamic acidresidue of the peptidoglycan stem peptide, formation of a minormuropeptide with L-alanine instead of glycine at position 1 of theinterpeptide bridge, perform a yet unknown function, or are involved inan early step of peptidoglycan precursors biosynthesis (addition ofL-lysine), respectively.

Thus far each new antibiotic gives rise to resistance strains, emergethat are resistance to multiple drugs and increasingly persistent formsof resistance begin to emerge. Drug resistance of S. aureus infectionsalready poses significant treatment difficulties, which are likely toget much worse unless new therapeutic agents are developed. Since S.aureus is likely involved in the synthesis of the peptidoglycan crossbridges in S. aureus, the gene provides an important tool in studyingthe mechanisms of antibiotic resistance. The S. aureus gene and itspolypeptides are also potential target for antagonists or agonists,which may be useful as antibiotics, or useful to block resistance toother antibiotics. That is, antagonists or agonists, such as smallmolecules, may be useful as antibiotics themselves, act additively withother antibiotics, or act synergistically with other antibiotics.

BRIEF SUMMARY OF THE INVENTION

The present invention provides isolated S. aureus polynucleotides andpolypeptides shown in Table 1 and SEQ ID NO:1 through SEQ ID NO:22(polynucleotide sequences having odd SEQ ID NOs and polypeptidesequences having even SEQ ID NOs). One aspect of the invention providesisolated nucleic acid molecules comprising polynucleotides having anucleotide sequence selected from the group consisting of: (a) anucleotide sequence shown in Table 1; (b) a nucleotide sequence encodingany of the amino acid sequences of the polypeptides shown in Table 1;and (c) a nucleotide sequence complementary to any of the nucleotidesequences in (a) or (b). The invention further provides for fragments ofthe nucleic acid molecules of (a), (b) & (c) above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical, to any of the nucleotide sequences in (a), (b) or (c)above, or a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide in (a), (b) or (c) above.Additional nucleic acid embodiments of the invention relate to isolatednucleic acid molecules comprising polynucleotides which encode the aminoacid sequences of epitope-bearing portions of a S. aureus polypeptidehaving an amino acid sequence in (a) above.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells. The present invention furtherrelates to the use of these vectors in the production of S. aureuspolypeptides or peptides by recombinant techniques.

The invention further provides isolated S. aureus polypeptides having anamino acid sequence selected from the group consisting of an amino acidsequence of any of the polypeptides described in Table 1 or fragmentsthereof.

The polypeptides of the present invention also include polypeptideshaving an amino acid sequence with at least 70% similarity, and morepreferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%similarity to those described in Table 1, as well as polypeptides havingan amino acid sequence at least 70% identical, more preferably at least75% identical, and still more preferably 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to those above; as well as isolated nucleic acidmolecules encoding such polypeptides.

The present invention further provides a vaccine, preferably amulti-component vaccine comprising one or more of the S. aureuspolynucleotides or polypeptides described in Table 1, or fragmentsthereof, together with a pharmaceutically acceptable diluent, carrier,or excipient, wherein the S. aureus polypeptide(s) are present in anamount effective to elicit an immune response to members of theStaphylococcus genus, or at least S. aureus, in an animal. The S. aureuspolypeptides of the present invention may further be combined with oneor more immunogens of one or more other staphylococcal ornon-staphylococcal organisms to produce a multi-component vaccineintended to elicit an immunological response against members of theStaphylococcus genus and, optionally, one or more non-staphylococcalorganisms.

The vaccines of the present invention can be administered in a DNA form,e.g., “naked” DNA, wherein the DNA encodes one or more staphylococcalpolypeptides and, optionally, one or more polypeptides of anon-staphylococcal organism. The DNA encoding one or more polypeptidesmay be constructed such that these polypeptides are expressed as fusionproteins.

The vaccines of the present invention may also be administered as acomponent of a genetically engineered organism or host cell. Thus, agenetically engineered organism or host cell which expresses one or moreS. aureus polypeptides may be administered to an animal. For example,such a genetically engineered organism or host cell may contain one ormore S. aureus polypeptides of the present invention intracellularly, onits cell surface, or in its periplasmic space. Further, such agenetically engineered organism or host cell may secrete one or more S.aureus polypeptides. The vaccines of the present invention may also beco-administered to an animal with an immune system modulator (e.g., CD86and GM-CSF).

The invention also provides a method of inducing an immunologicalresponse in an animal to one or more members of the Staphylococcusgenus, preferably one or more isolates of the S. aureus species,comprising administering to the animal a vaccine as described above.

The invention further provides a method of inducing a protective immuneresponse in an animal, sufficient to prevent, attenuate, or control aninfection by members of the Staphylococcus genus, preferably at least S.aureus species, comprising administering to the animal a compositioncomprising one or more of the polynucleotides or polypeptides describedin Table 1, or fragments thereof. Further, these polypeptides, orfragments thereof, may be conjugated to another immunogen and/oradministered in admixture with an adjuvant.

The invention further relates to antibodies elicited in an animal by theadministration of one or more S. aureus polypeptides of the presentinvention and to methods for producing such antibodies and fragmentsthereof. The invention further relates to recombinant antibodies andfragments thereof and to methods for producing such antibodies andfragments thereof.

The invention also provides diagnostic methods for detecting theexpression of the polynucleotides of Table 1 by members of theStaphylococcus genus in an animal. One such method involves assaying forthe expression of a polynucleotide encoding S. aureus polypeptides in asample from an animal. This expression may be assayed either directly(e.g., by assaying polypeptide levels using antibodies elicited inresponse to amino acid sequences described in Table 1) or indirectly(e.g., by assaying for antibodies having specificity for amino acidsequences described in Table 1). The expression of polynucleotides canalso be assayed by detecting the nucleic acids of Table 1. An example ofsuch a method involves the use of the polymerase chain reaction (PCR) toamplify and detect Staphylococcus nucleic acid sequences.

The present invention also relates to nucleic acid probes having all orpart of a nucleotide sequence described in Table 1 (odd SEQ ID NOs)which are capable of hybridizing under stringent conditions toStaphylococcus nucleic acids. The invention further relates to a methodof detecting one or more Staphylococcus nucleic acids in a biologicalsample obtained from an animal, said one or more nucleic acids encodingStaphylococcus polypeptides, comprising: (a) contacting the sample withone or more of the above-described nucleic acid probes, under conditionssuch that hybridization occurs, and (b) detecting hybridization of saidone or more probes to the Staphylococcus nucleic acid present in thebiological sample.

Polynucleotides and Polypeptides of the Invention

Features of femX Polynucleotides and Polypeptides.

The nucleotide sequence shown in SEQ ID NO:1 was determined bysequencing the S. aureus overlapping clones BTEFS71 and BTEJE39. Thenucleotide sequence contains an open reading frame encoding the femXpolypeptide comprising 414 amino acid residues (SEQ ID NO:2), includingan initiation codon encoding an N-terminal methionine at nucleotidepositions 164-166, and a predicted molecular weight of about 49.1 kDa.

The femX polypeptides of the present invention have amino acid sequencehomology to known genes involved in formation of peptidoglycan crossbridges, including the conserved cysteine pattern characteristic of theepr and fem family of genes. The S. aureus femX polypeptide of SEQ IDNO:2 was found to share a high degree of local sequence identity withamino acid sequences of the epr (M. Sugai, et al. (1997) J. Bacteriol.179(13):4311-4318) and fem A and fem B proteins from Staphlococcusspecies (A. M. Stranden et al., (1997) J. Bacteriol. 179(1):9-16; G.Thumm et al. (1997) Mol. Microbiol. 23(6):1251-1265; W. E. Alborn etal., (1996) Gene 180(1-2):177-181) using the computer program BLAST(Altschul et al., (1990) J. Mol. Biol. 215:403-410).

The strong homology between species and identity among fem proteins ofS. aureus indicates that femX is involved in peptidoglycan interpeptidebridge biosynthesis. Thus, the polypeptides of the present invention areuseful in screening methods to make antagonists which block theirfunction. Antagonists can be used, for instance, as antibiotics to treatantibiotic resistant S. aureus or other Staphylococcus species.Antagonists of the polypeptides of the present invention can beidentified by measuring the formation of peptidoglycan cross bridges.More specifically, the synthesis of glycines 1-5 of peptidoglycan crossbridges can be measured as exemplified by A. M. Stranden et al. (1997)J. Bacteriol 179(1):9-16 (incorporated herein in its entirety).Antagonists of femX would act to inhibit peptidoglycan cross bridgeformation.

Other uses of the femX polypeptides of the present invention include:inter alia, to detect S. aureus in immunoassays, as epitope tags, asmolecular weight markers on SDS-PAGE gels, as molecular weight markersfor molecular sieve gel filtration columns, to generate antibodies thatspecifically bind S. aureus femX for the detection S. aureus inimmunoassays, to generate an immune response against S. aureus and otherStaphylococcus species, and as vaccines against S. aureus and otherStaphylococcus species.

Isolated nucleic acid molecules of the present invention, particularlyDNA molecules, are useful as probes for gene mapping and for identifyingS. aureus in a biological samples, for instance, by Southern andNorthern blot analysis. femX polynucleotides of the present inventionare also useful in detecting S. aureus by PCR using primers for femXpolynucleotides. Isolated polynucleotides of the present invention arealso useful in making the polypeptides of the present invention.

Features of furA, furB, and furC Polynucleotides and Polypeptides.

The nucleotide sequences for furA, furB, and furC were determined bysequencing the S. aureus clones BTEJQ50 (SEQ ID NO:3), BTALE70 (SEQ IDNO:5), and BTEBP80 and BTEFD68 (collectively SEQ ID NO:7). Thenucleotide sequence of SEQ ID NO:3 contains an open reading frameencoding the furA polypeptide comprising 136 amino acid residues (SEQ IDNO:4), including an initiation codon encoding an N-terminal methionineat nucleotide positions 101-103, and a predicted molecular weight ofabout 15.9 kDa.

The nucleotide sequence of SEQ ID NO:5 contains an open reading frameencoding the furB polypeptide comprising 148 amino acid residues (SEQ IDNO:6), including an initiation codon encoding an N-terminal methionineat nucleotide positions 101-103, and a predicted molecular weight ofabout 17.2 kDa.

The nucleotide sequence of SEQ ID NO:7 contains an open reading frameencoding the furC polypeptide comprising 149 amino acid residues (SEQ IDNO:8), including an initiation codon encoding an N-terminal leucine atnucleotide positions 101-103, and a predicted molecular weight of about17.2 kDa.

The fur (ferric uptake regulator) polypeptides (furA, furB, and furC) ofthe present invention have amino acid sequence homology to known genesinvolved in iron regulation. The S. aureus furA polypeptide of SEQ IDNO:2 was found to share a high degree of local sequence identity withthe amino acid sequence of a fur gene from Staphylococcus epidermidis(GenBank accession number gnl|PID|e236389). See C. Heidrich et al.(1996) FEMS Micro. Letts. 140:253-259. The S. aureus furB polypeptide ofSEQ ID NO:2 was found to share a high degree of local sequence identitywith the amino acid sequence of a fur family gene from Bacillus subtilis(GenBank accession number gnl|PID|e281583). See N. J Cummings et al.(1997) Microbiology 143:1855-1859. The S. aureus furC polypeptide of SEQID NO:2 was found to share a high degree of local sequence identity withthe amino acid sequence of another fur family gene from Bacillussubtilis (GenBank accession number gnl|PID|e1185621). See F. Kunst etal. (1997) Nature 390:249-256.

The fur polypeptides of the present invention also share identity amongthemselves as well as other fur and fur-like genes from Bacillussubtilis (GenBank accession numbers gnl|PID|e1185777), Streptococcuspyogenes (GenBank accession number gi|1667516), Neisseria meningitidis(GenBank accession number gi|433299), Neisseria gonorrheae (GenBankaccession number gi|349012), Camplyobacter upsaliensis (GenBankaccession number gi|1228779), Camplyobacter jejuni (GenBank accessionnumber gi|511113) Mycobacterium tuberculosis (GenBank accessionnumbergnl|PID|e315163), and other bacteria species. Identities werecompared using the computer program BLAST (Altschul et al., (1990) J.Mol. Biol. 215:403-410).

The strong homology among the fur proteins of S. aureus and otherbacteria species indicates that furA, furB, and furC are involved iniron regulation in S. aureus. Since iron is essential for the growth andmultiplication of nearly microorganisms, the polypeptides of the presentinvention are useful in screening methods to make antagonists whichblock their function. Antagonists can be used, for instance, asantibiotics to treat infections of S. aureus or other Staphylococcusspecies. Antagonists of the polypeptides of the present invention can beidentified by measuring the ability of bacteria to grow in the presenceof varying concentrations of iron.

Other uses of the fur polypeptides of the present invention include:inter alia, to detect S. aureus in immunoassays, as epitope tags, asmolecular weight markers on SDS-PAGE gels, as molecular weight markersfor molecular sieve gel filtration columns, to generate antibodies thatspecifically bind S. aureus furA, furB, and furC for the detection S.aureus in immunoassays, to generate an immune response against S. aureusand other Staphylococcus species, and as vaccines against S. aureus,other Staphylococcus species and other bacteria genuses.

Isolated nucleic acid molecules of the present invention, particularlyDNA molecules, are useful as probes for gene mapping and for identifyingS. aureus in a biological samples, for instance, by Southern andNorthern blot analysis. fur polynucleotides of the present invention arealso useful in detecting S. aureus by PCR using primers for a particularfur polynucleotide. Isolated polynucleotides of the present inventionare also useful in making the polypeptides of the present invention.

Features of fmtB, pbpF, and pbpG Polynucleotides and Polypeptides.

The nucleotide sequences for fmtB, pbpF, and pbpG were determined bysequencing the S. aureus clones BTEDA22 and BTEDV18 (SEQ ID NO:9),BTEBG73 and BTAJO70 (SEQ ID NO:11), and BTEBU53 and BTEFB55 (SEQ IDNO:13), respectively. The nucleotide sequence of SEQ ID NO:9 contains anopen reading frame encoding the fmtB polypeptide comprising 498 aminoacid residues (SEQ ID NO:10), including an initiation codon encoding anN-terminal methionine at nucleotide positions 101-103, and a predictedmolecular weight of about 56.4 kDa.

The nucleotide sequence of SEQ ID NO:11 contains an open reading frameencoding the pbpF polypeptide comprising 691 amino acid residues (SEQ IDNO:12), including an initiation codon encoding an N-terminal leucine atnucleotide positions 101-103, and a predicted molecular weight of about77.2 kDa.

The nucleotide sequence of SEQ ID NO:13 contains an open reading frameencoding the pbpG polypeptide comprising 301 amino acid residues (SEQ IDNO:14), including an initiation codon encoding an N-terminal methionineat nucleotide positions 101-103, and a predicted molecular weight ofabout 34.5 kDa.

The fmtB, pbpF, and pbpG polypeptides of the present invention haveamino acid sequence homology to known penicillin-biding proteins amongseveral species. The S. aureus fmtB polypeptide of SEQ ID NO:10 wasfound to share local sequence identity, inter alia, with the amino acidsequence of a penicillin-binding protein gene from Bacillus subtilis(GenBank accession number gnl|PID|e1185286). See F. Kunst et al. (1997)Nature 390:249-256. fmtB also shares sequence identity with anotherStaphylococcus aureus polypeptide associated with antibiotic resistance(GenBank accession number gnl|PID|d1024918). See H. Komatsuzawa et al.(1997) Antimicrob. Agents Chemother. 41:2355-2361.

The S. aureus pbpF polypeptide of SEQ ID NO:12 was found to share localsequence identity, inter alia, with the amino acid sequence ofpenicillin-binding genes from Bacillus subtilis (GenBank accessionnumber gnl|PID|e1181903 and gnl|PID|e185767) and Streptococcusthermophilus (GenBank accession number gi|643510). The S. aureus pbpGpolypeptide of SEQ ID NO:14 was found to share local sequence identity,inter alia, with the amino acid sequence of penicillin-binding genesfrom Pseudomonas syringae (GenBank accession number gi|551940). See E.Roine et al. (1996) J. Bacteriol. 178:410-417, and Bacillus subtilis(GenBank accession number gnl|PID|e267588). Identities were comparedusing the computer program BLAST (Altschul et al, (1990) J. Mol. Biol.215:403-410).

The strong homology among the S. aureus fmtB, pbpF, and pbpGpolypeptides of the present invention and penicillin-binding proteinsfrom other bacteria species indicates that fmtB, pbpF, and pbpG areinvolved cell wall synthesis and in β-lactam resistance in S. aureus.The polypeptides of the present invention are therefore useful formaking compounds that inhibit their function for use as antibiotics.Inhibitors of the polypeptides of the present invention can beidentified by measuring the ability of bacteria to grow in the presenceof varying concentrations of antibiotics or by cell wall synthesisassays using methods known in the art.

Other uses of the fmtB, pbpF, and pbpG polypeptides of the presentinvention include: inter alia, to detect S. aureus in immunoassays, asepitope tags, as molecular weight markers on SDS-PAGE gels, as molecularweight markers for molecular sieve gel filtration columns, to generateantibodies that specifically bind S. aureus fmtB, pbpF, or pbpG for thedetection S. aureus in immunoassays, to generate an immune responseagainst S. aureus and other Staphylococcus species, and as vaccinesagainst S. aureus, other Staphylococcus species and other bacteriagenuses.

Isolated nucleic acid molecules of the present invention, particularlyDNA molecules, are useful as probes for gene mapping and for identifyingS. aureus in a biological samples, for instance, by Southern andNorthern blot analysis. fmtB, pbpF, and pbpG polynucleotides of thepresent invention are also useful in detecting S. aureus by PCR usingprimers for a particular fmtB, pbpF, or pbpG polynucleotide. Isolatedpolynucleotides of the present invention are also useful in making thepolypeptides of the present invention.

Features of cbrA, cbrB, and cbrC Polynucleotides and Polypeptides.

The nucleotide sequences for cbrA (SEQ ID NO:15), cbrB (SEQ ID NO:17),and cbrC (SEQ ID NO:19) comprise a single operon and were determined bysequencing the S. aureus overlapping clones BTACA44 and BTAGJ54 whichspan the operon. The nucleotide sequence of SEQ ID NO:15 contains anopen reading frame encoding the cbrA polypeptide comprising 330 aminoacid residues (SEQ ID NO:16), including an initiation codon encoding anN-terminal methionine at nucleotide positions 7-9, and a predictedmolecular weight of about 36.8 kDa.

The nucleotide sequence of SEQ ID NO:17 contains an open reading frameencoding the cbrB polypeptide comprising 331 amino acid residues (SEQ IDNO:18), including an initiation codon encoding an N-terminal leucine atnucleotide positions 19-21, and a predicted molecular weight of about35.5 kDa.

The nucleotide sequence of SEQ ID NO:19 contains an open reading frameencoding the cbrC polypeptide comprising 332 amino acid residues (SEQ IDNO:20), including an initiation codon encoding an N-terminal methionineat nucleotide positions 91-93, and a predicted molecular weight of about35.7 kDa.

The cbr polypeptides (cbrA, cbrB, and cbrC) of the present inventionhave amino acid sequence homology to known genes involved in ironregulation. The S. aureus cbrA (SEQ ID NO:16), cbrB (SEQ ID NO:18), andcbrC (SEQ ID NO:20) polypeptides were found to share local sequenceidentity among themselves and with the amino acid sequence of a cbrA,cbrB, and cbrC genes from Erwinia chrysanthemi (GenBank accessionnumbers gi|809541, gi|809542, and gi|809541 respectively). See B. Maheet al. (1995) Mol. Microbiol. 18:33-43. The cbrA, cbrB, and cbrCpolypeptides of the present invention also share sequence identity andwith iron regulatory genes of other bacterial species including Bacillussubtilis (GenBank accession number gnl|PID|e1182834, gnl|PID|e1182835,and gnl|PID|e1182836). See F. Kunst et al. (1997) Nature 390:249-256 andBacillus intermedius (GenBank accession number gnl|PID|e245932).Identities were compared using the computer program BLAST (Altschul etal., (1990) J. Mol. Biol. 215:403-410).

The strong homology among the cbr proteins of S. aureus and otherbacteria species indicates that cbrA, cbrB, and cbrC are involved iniron regulation in S. aureus. Since iron is essential for the growth andmultiplication of nearly microorganisms, the polypeptides of the presentinvention are useful in screening methods to make antagonists whichblock their function. Antagonists can be used, for instance, asantibiotics to treat infections of S. aureus or other Staphylococcusspecies. Antagonists of the polypeptides of the present invention can beidentified by measuring the ability of bacteria to grow in the presenceof varying concentrations of iron.

Other uses of the polypeptides of the present invention include: interalia, to detect S. aureus in immunoassays, as epitope tags, as molecularweight markers on SDS-PAGE gels, as molecular weight markers formolecular sieve gel filtration columns, to generate antibodies thatspecifically bind S. aureus polypeptides of the present invention forthe detection S. aureus in immunoassays, to generate an immune responseagainst S. aureus and other Staphylococcus species, and as vaccinesagainst S. aureus, other Staphylococcus species and other bacteriagenuses.

Isolated nucleic acid molecules of the present invention, particularlyDNA molecules, are useful as probes for gene mapping and for identifyingS. aureus in a biological samples, for instance, by Southern andNorthern blot analysis. S. aureus polynucleotides of the presentinvention are also useful in detecting S. aureus by PCR using primersfor a particular S. aureus polynucleotide. Isolated polynucleotides ofthe present invention are also useful in making the polypeptides of thepresent invention.

Features of Enolase Polynucleotides and Polypeptides.

The nucleotide sequence shown in SEQ ID NO:21 was determined bysequencing the S. aureus overlapping clones BTAAI44 and BTAGE12. Thenucleotide sequence contains an open reading frame encoding the enolasepolypeptide comprising 434 amino acid residues (SEQ ID NO:22), includingan initiation codon encoding an N-terminal methionine at nucleotidepositions 103-105, and a predicted molecular weight of about 47.1 kDa.

The enolase polypeptides of the present invention have amino acidsequence identity homology to known enolase genes from other bacterialspecies. The S. aureus enolase polypeptide of SEQ ID NO:22 shares localsequence identity with amino acid sequences of the enolase genes fromother bacterial species including Bacillus subtilis (GenBank accessionnumbers gi|460259 and gnl|PID|e1186078), Spongilla sp. (GenBankaccession number gi|1839206), Mycobacterium tuberculosis (GenBankaccession number gnl|PID|e304557) Methanococcus jannaschii (GenBankaccession number gi|1590967) and Campylobacter jejuni (GenBank accessionnumber gi|437277).

The S. aureus enolase protein of the present invention was identified asa molecule involved in laminin (LN)/laminin receptor (LNRec)interactions. The S. aureus enolase protein of the present invention wasshown to be responsible for LNRec activity in bridging experimentsbetween the S. aureus and MDCK cell in culture. The S. aureus enolasegene of the present invention was cloned by first generating monoclonalantibodies against the LNRec molecule and using the antibodies tosubsequently isolated the LNRec molecule. The LNRec molecule waspurified and partially sequenced. Partial amino acid sequence analysiswas used to clone and isolate the S. aureus enolase gene of the presentinvention. A characteristic feature of infection by S. aureus isbloodstream invasion and widespread metastatic abscess formation. The S.aureus enolase polypeptides of the present invention therefore representa target for both vaccines and antibiotics. Antibiotics of the presentinvention include peptides, polypeptides, antibodies (and fragmentsthereof), small molecules, and other drugs that bind the S. aureusenolase polypeptides of the present invention, or enolase associatedmolecules, and prevent binding of S. aureus to laminin. The blockingmolecules of the present invention may act by directly blocking thebinding of enolase polypeptides to laminin or enolase associatedmolecules to laminin. Assays for measuring the binding of molecules toenolase polypeptides or enolase associated molecules; assays formeasuring the binding of S. aureus to laminin; and assays for measuringthe metastatic activity of S. aureus include those described andreferenced in Lopes et al. (1985) Science 229:275-277, described andreferenced in Brentani (1989) Oncogenesis 1:247-260, known in the art,and disclosed herein.

The structural homology and identity between the enolase polypeptides ofS. aureus and those of other bacterial species indicates that theenolase polypeptides of S. aureus share the same function as the enolasepolypeptides from other bacterial enolase polypeptides, including thosedescribed by Babbitt et al. (1996) Biochemistry 35:16489-16501. Theenolase polynucleotides and polypeptides of the present invention areuseful to produce mutant S. aureus enolase genes, polypeptides, andmutant S. aureus strains for use as vaccines and to induce an immuneresponse in humans and other animals by using the methods described inU.S. Pat. Nos. 5,703,219 and 5,703,219. In the methods of U.S. Pat. Nos.5,703,219 and 5,703,219 the S. aureus enolase polypeptides andpolypeptides are substituted for the Helicobacter pyiori enolasepolypeptides and polypeptides. Modifications to accommodate the S.aureus enolase polypeptides and polypeptides of the present inventionare made using information disclosed herein and known in the art.

Other uses of the enolase polypeptides of the present invention include:inter alia, to detect S. aureus in immunoassays, as epitope tags, asmolecular weight markers on SDS-PAGE gels, as molecular weight markersfor molecular sieve gel filtration columns, to generate antibodies thatspecifically bind S. aureus enolase for the detection S. aureus inimmunoassays, to generate an immune response against S. aureus and otherStaphylococcus species, and as vaccines against S. aureus and otherStaphylococcus species as discussed above.

Isolated nucleic acid molecules of the present invention, particularlyDNA molecules, are useful as probes for gene mapping and for identifyingS. aureus in a biological samples, for instance, by Southern andNorthern blot analysis. enolase polynucleotides of the present inventionare also useful in detecting S. aureus by PCR using primers for enolasepolynucleotides. Isolated polynucleotides of the present invention arealso useful in making the polypeptides of the present invention.

DETAILED DESCRIPTION

The present invention relates to recombinant S. aureus nucleic acids andfragments thereof. The present invention further relates to recombinantS. aureus polypeptides and fragments thereof. The invention also relatesto methods for using these polypeptides to produce immunologicalresponses and to confer immunological protection to disease caused bymembers of the genus Staphylococcus, at least isolates of the S. aureusgenus. The invention further relates to nucleic acid sequences whichencode antigenic S. aureus polypeptides and to methods for detecting S.aureus nucleic acids and polypeptides in biological samples. Theinvention also relates to antibodies specific for the polypeptides andpeptides of the present invention and methods for detecting suchantibodies produced in a host animal.

Definitions

The following definitions are provided to clarify the subject matterwhich the inventors consider to be the present invention.

As used herein, the phrase “pathogenic agent” means an agent whichcauses a disease state or affliction in an animal. Included within thisdefinition, for examples, are bacteria, protozoans, fungi, viruses andmetazoan parasites which either produce a disease state or render ananimal infected with such an organism susceptible to a disease state(e.g., a secondary infection). Further included are species and strainsof the genus Staphylococcus which produce disease states in animals.

As used herein, the term “organism” means any living biological system,including viruses, regardless of whether it is a pathogenic agent.

As used herein, the term “Staphylococcus” means any species or strain ofbacteria which is members of the genus Staphylococcus. Such species andstrains are known to those of skill in the art, and include those thatare pathogenic and those that are not.

As used herein, the phrase “one or more S. aureus polypeptides of thepresent invention” means polypeptides comprising the amino acid sequenceof one or more of the S. aureus polypeptides described in Table 1 (evenSEQ ID NOs). These polypeptides may be expressed as fusion proteinswherein the S. aureus polypeptides of the present invention are linkedto additional amino acid sequences which may be of staphylococcal ornon-staphylococcal origin. This phrase further includes polypeptidecomprising fragments of the S. aureus polypeptides of the presentinvention. Additional definitions are provided throughout thespecification.

Explanation of Table 1

Table 1, below, shows the nucleotide sequence of 11 genes from S. aureusand the sequence of the polypeptides they encode. The table lists thename of the S. aureus gene, followed by a sequence identification number(SEQ ID NO:), and the gene's nucleotide or polypeptide sequence. Thetable also lists the plasmid clones comprising the nucleotide sequences.The actual nucleotide or amino acid sequence of each gene is also shownin the Sequence Listing under the corresponding SEQ ID NO.

Explanation of Table 2

Table 2 lists residues comprising antigenic epitopes of antigenicepitope-bearing fragments present in each of the S. aureus polypeptidesdescribed in Table 1 as predicted by the inventors using the algorithmof Jameson and Wolf, (1988) Comp. Appl. Biosci. 4:181-186. TheJameson-Wolf antigenic analysis was performed using the computer programPROTEAN (Version 3.11 for the Power Macintosh, DNASTAR, Inc., 1228 SouthPark Street Madison, Wis.). Each S. aureus polypeptide shown in Table 1has one or more antigenic epitopes comprising residues described inTable 2. It will be appreciated that depending on the analyticalcriteria used to predict antigenic determinants, the exact address ofthe determinant may vary slightly. The residues and locations showndescribed in Table 2 correspond to the amino acid sequences for eachgene shown in Table 1 and in the Sequence Listing.

Explanation of Table 3.

The S. aureus polypeptides of the present invention may include one ormore conservative amino acid substitutions from natural mutations orhuman manipulation as indicated in Table 3. Changes are preferably of aminor nature, such as conservative amino acid substitutions that do notsignificantly affect the folding or activity of the protein. Residuesfrom the following groups, as indicated in Table 3, may be substitutedfor one another: Aromatic, Hydrophobic, Polar, Basic, Acidic, and Small.

Nucleic Acid Molecules

Sequenced S. aureus genomic DNA was obtained from the S. aureus strainISP3. S. aureus strain ISP3, has been deposited at the American TypeCulture Collection, as a convenience to those of skill in the art. TheS. aureus strain ISP3 was deposited on Apr. 7, 1998 at the ATCC, 10801University Blvd. Manassas, Va. 20110-2209, and given accession number202108. As discussed elsewhere herein, polynucleotides of the presentinvention readily may be obtained by routine application of well knownand standard procedures for cloning and sequencing DNA. Detailed methodsfor obtaining libraries and for sequencing are provided below, forinstance. A wide variety of Staphylococcus aureus strains that can beused to prepare S aureus genomic DNA for cloning and for obtainingpolynucleotides and polypeptides of the present invention. A widevariety of Staphylococcus aureus strains are available to the publicfrom recognized depository institutions, such as the American TypeCulture Collection (ATCC). It is recognized that minor variation is thenucleic acid and amino acid sequence may be expected from S aureusstrain to strain. The present invention provides for genes, includingboth polynucleotides and polypeptides, of the present invention from allthe Staphylococcus aureus strains. That is, the femX, furA-C, fmtB, pbpGand -F, and CbrA-C genes from all Staphylococcus aureus strains areincluded in the present invention.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc., FosterCity, Calif.), and all amino acid sequences of polypeptides encoded byDNA molecules determined herein were predicted by translation of a DNAsequence determined as above. Therefore, as is known in the art for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion. In case of conflict between Table 1 and either the nucleicacid sequence of the clones listed in Table 1 or the amino acid sequenceof the protein expressed by the clones listed in Table 1, the cloneslisted in Table 1 are controlling. By “nucleotide sequence” of a nucleicacid molecule or polynucleotide is intended to mean either a DNA or RNAsequence. Using the information provided herein, such as the nucleotidesequence in Table 1, a nucleic acid molecule of the present inventionencoding a S. aureus polypeptide may be obtained using standard cloningand screening procedures, such as those for cloning DNAs using genomicDNA as starting material. See, e.g., Sambrook et al. MOLECULAR CLONING:A LABORATORY MANUAL (Cold Spring Harbor, N.Y. 2nd ed. 1989); Ausubel etal., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley and Sons, N.Y.1989).

TABLE 1 Nucleotide and Amino Acid Sequences of 11 S. aureus Genes. femXnucleotide sequence clones BTEFS71 and BTEJE39TGGAAAATTAATGAAGTTCCAAAGTTTAGATCAAAACTGGAATAATGGTGGAT (SEQ ID NO:1)GGCGTAAAGCAGAGGTTGCACATAAAGTTGTTCATAATTATGAAAATGATATGATTTTTATTAGACCATTTAAAAAAGCATAATTTAAATCGAAGGCAGGACATTGAAATATGAAATTTTCAACTTTAAGTGAAGAAGAATTTACCAACTACACCAAAAAGCACTTCAAACATTATACGCAGTCTATAGAATTATATAATTATAGAAATAAAATAAATCATGAAGCACATATTGTGGGAGTGAAGAATGATAAAAATGAAGTTATAGCTGCATGTTTATTAACAGAGGCACGAATTTTTAAATTCTACAAATATTTCTACTCTCATAGAGGTCCTTTACTTGATTATTTCGATGCTAAATTAGTTTGTTACTTTTTTAAAGAATTATCTAAATTCATTTATAAAAATAGAGGAGTATTTATTCTTGTTGATCCATATTTAATAGAGAATTTAAGAGATGCAAATGGTAGGATAATAAAGAATTATAATAATTCAGTGATAGTAAAGATGCTAGGGAAAATTGGGTATCTCCATCAAGGTTATACAACAGGATATTCAAATAAAAGTCAAATTAGGTGGATTTCTGTATTGGATTTAAAAGATAAAGATGAGAATCAACTTTTAAAAGAAATGGAATACCAAACTAGAAGAAATATAAAAAAGACTATTGAGATTGGTGTTAAGGTTGAAGATTTATCTATTGAAGAAACAAATCGATTTTATAAATTGTTTCAAATGGCTGAAGAAAAACATGGTTTTCATTTCATGAATGAAGATTATTTTAAACGAATGCAAGAAATATATAAAGATAAGGCAATGTTAAAGATAGCTTGTATAAATCTTAATGAATATCAAGATAAATTAAAAATACAATTATTGAAAATCGAAAATGAAATGATGACTGTGAACAGAGCATTAAATGAAAATCCAAATTCTAAAAAAAATAAATCAAAATTAAATCAGTTAAATATGCAATTATCTAGTATTAATAATAGAATTAGTAAAACCGAAGAACTAATATTTGAAGATGGACCTGTTTTGGATTTAGCTGCTGCTTTATTTATATGTACTGATGATGAAGTTTATTATCTATCAAGTGGATCAAATCCGAAATATAATCAGTATATGGGTGCATATCATCTACAATGGCATATGATAAAATATGCAAAATCACATAATATTAATAGGTATAATTTTTATGGAATAACAGGCGTCTTTAGTAATGAGGCGGATGATTTTGGTGTTCAACAATTTAAAAAGGGTTTTAATGCACATGTTGAAGAATTAATTGGTGATTTCATCAAACCAGTAAGACCAATTCTATATAAATTTGCAAAACTTATTTATAAGGTTTAATTATAAAGTATGTTGGAAATTGAAATTTTAAATTCTTTCCAACATACTTTTCACTTTTTAAG femX amino acid sequence clonesBTEFS71 and BTEJE39MKFSTLSEEEFTNYTKKHFKHYTQSIELYNYRNKINHEAHIVGVKNDKNEVIAACL (SEQ ID NO:2)LTEARIFKFYKYFYSHRGPLLDYFDAKLVCYFFKELSKFIYKNRGVFILVDPYLIENLRDANGRIIKNYNNSVIVKMLGKIGYLHQGYTTGYSNKSQIRWISVLDLKDKDENQLLKEMEYQTRRNIKKTIEIGVKVEDLSIEETNRFYKLFQMAEEKHGFHFMNEDYFKRMQEIYKDKAMLKIACINLNEYQDKLKIQLLKIENEMMTVNRALNENPNSKKNKSKLNQLNMQLSSINNRISKTEELIFEDGPVLDLAAALFICTDDEVYYLSSGSNPKYNQYMGAYHLQWHMIKYAKSHNINRYNFYGITGVFSNEADDFGVQQFKKGFNAHVEELIGDFIKPVRPILYKFAKLIYKV furA nucleotide sequence clone: BTEJQ50TCTCCGGGTGGTGAaATTGTAGTTCTACTTGTTATTTTACTTATGATTACAATGG (SEQ ID NO:3)CTTATCAGAAAATGCGAATGAAGTTTAAAAAGGGAGCTAATATCAATGAATACAAATGATGCTATTAAAATTTTAAAAGAGAACGGTTTAAAATATACAGATAAACGTAAAGATATGTTAGATATTTTTGTCGAAGAAGATAAGTATATAAACGCAAAGTATATACAACAAGTTATGGATGAAAATTATCCTGGAATTTCATTCGACACAATATATAGAAACCTGCACTTATTTAAAGATTTAGGAATTATTGAAAATACAGAACTTGATGGTGAAATGAAGTTTAGAATCGCTTGTACAAACCATCATCATCATCATTTTATCTGTGAAAAGTGTGGAGATACAAAGGTAATAGATTATTGTCCAATAGATCAGATAAAATTATCACTACCTGGTGTTAATATTCACAAACACAAACTTGAAGTTTATGGTGTATGTGAGTCTTGCCAAGATTAATATAAAGAAATGAGATTTATGCACATTTGGTCCGATGTATGCATAAATCT furA amino acid sequence clone: BTEJQ50MNTNDAIKILKENGLKYTDKRKDMLDIFVEEDKYINAKYIQQVMDENYPGISFDTI (SEQ ID NO:4)YRNLHLFKDLGIIENTELDGEMKFRIACTNHHHHHFICEKCGDTKVIDYCPIDQIKLSLPGVNIHKHKLEVYGVCESCQD furB nucleotide sequence clone: BTALE70TTAAATGAAATCATCATGTAAATATTGACACGCGCGCAATACTACAGTTATAT (SEQ ID NO:5)TTATAGTAAGTAATAATAATTATTATATAAGAAAGATGGTGATATAGATGAGTGTTGAAATAGAATCAATTGAACATGAACTAGAAGAATCAATTGCATCATTGCGACAAGCAGGCGTAAGAATTACACCTCAAAGACAAGCAATATTACGTTATTTAATTTCTTCACATACTCATCCAACAGCTGATGAAATTTATCAAGCACTTTCACCTGATTTTCCAAATATAAGTGTTGCGACAATATATAATAACTTAAGAGTGTTTAAAGATATTGGAATTGTAAAAGAATTAACATATGGAGACTCATCAAGTCGATTCGACTTTAATACACATAATCATTATCATATTATATGTGAACAATGTGGTAAGATTGTTGATTTTCAATATCCACAGTTAAATGAAATTGAAAGATTAGCTCAGCATATGACTGACTTTGACGTAACACATCATCGAATGGAAATTTATGGAGTTTGTAAAGAATGCCAAGATAAATAATTTAACTTTGGTAGTATGACAAATTAAAAAAGCGTTAC T furB amino acidsequence clone: BTALE70MSVEIESIEHELEESIASLRQAGVRITPQRQAILRYLISSHTHPTADEIYQALSPDFPNI (SEQ IDNO:6) SVATTYNNLRVFKDIGIVKELTYGDSSSRFDFNTHNHYHIICEQCGKIVDFQYPQLNEIERLAQHMTDFDVTHHRMEIYGVCKECQDK furC nucleotide sequence clones: BTEBP80and BTEFD68 TGAGAAAAGCTTGCATTTTATTGAGAAAACTGTTAGTTTTAATTGTAAAGTTTG (SEQID NO:7) AAATAATTTGTAATGATTTTAATTATTAGTAGGGGAGTGGACATCGTTGGAAGAACGATTAAATCGCGTTAAGCAACAATTACAACAATCATCATATAAGCTAACGCCACAACGCGAAGCTACTGTTAGAGTTCTAATTGAAAATGAAAAAGATCATCTAAGTGCTGAAGACGTATATCTGAAAGTAAAAGATAAAGCGCCTGAAATTGGCTTGGCGACAGTATACAGAACGTTAGAGTTGTTAGCTGAACTAAAAGTTGTCGACAAAATTAACTTTGGTGATGGCGTCGCTCGTTTTGATTTAAGAAAAGAAGGCGCAAAACATTTCCACCATCATTTAGTATGTATGGAATGTGGTCGTGTAGATGAAATCGATGAAGATTTGTTACCAGAAGTTGAAAATCGAGTTGAAAATGAGTTCAATTTTAAAATTTTAGATCATCGTTTAACTTTCCATGGTGTGTGTGAAACGTGCCAAGCTAAAGGTAAAGGATAGTAAATTGCGTAGGTTAAATTAACCTTCGCTTTTT TTAGAGGTGTGGTTATfurC amino acid sequence clones: BTEBP80 and BTEFD68LEELNRVKQQLQQSSYKLTPQREATVRVLIENEKDHLSAEDVYLKVKDKAPEIG (SEQ ID NO:8)LATVYRTLELLAELKVVDKINFGDGVARFDLRKEGAKHFHHHLVCMECGRVDEIDEDLLPEVENRVENEFNFKILDHRLTFHGVCETCQAKGKG fmtB nucleotide sequence clone:BTEDA22 and BTEDV18GTAAATATACCTCTTTAATTAATTTATTCAATAGAACTGGTATAATAAAATAA (SEQ ID NO:9)ATCTCATTAGGCACTTAAGTAAATTTAACATATAAAAAGGAACGTTTATGACTACTAAAAAACTGTATTTTCTATCCATTTCTATTATCATTTTAGTCGCCATTTCAATTGCTATATATATAACATTAAATAGCAATACGAAGACACGGTTAACCAATGATTCGCAACAACAAATAGATACAATTATCGAGCATGATTTACAAAAGGGACACATTCCTGGAGCATCAATTTTAATAGTAAAAAATGGCAAAGTTTTTTTAAATAAAGGTTATGGTTATCAAGATGTTGATAAAAAAGTCAAAGCTTCTCCCACAACAAAGTATGAAATTGCTTCTAATACGAAAGCTTTCACAGGTCTTGCAATTTTAAAATTAGCTCAAGAAGGTCGATTAAACTTAAATGATGCCGTATCCAAACATGTGCCTCATTTTAAAATGAACTATAATGGTCAAAATGAAACTATTACGATTAAGCAACTTTTGGCTCAAACAAGTGGTATACCTAGTGATATTACAAGCGAAGATTCTGTGACAAGCAAAAATAATCGTTTAAATGATGTAACCCATGCAATTATGGGTGATGAATTACATCATAAGCCCGGAGAAGAATTTGAATACTCAAATATGAACTATGATTTATTAGGTTTAATTATCCAAAACGTTACGAAGCAATCCTATACAAAATATATTACAAATTCATGGCTCAAGCCTTTGCATATGACACATACATCATTCAAACAAACCAATTACAAATCAAAACATGATGCTATTGGCTATGAATTACAAGGTTCGACACCTGTCGTCTCTAAACCTGAATTTAACCTTTGGGATACACCATCAGCATATATGATGACATCAACTGAAGATTTGGAACATTGGATAAAATTCCAACTTAATCCACCTGATAAATACAAATCATTAGTTCAACAATCACATAAAAATTTATCTTCAACAATTGGTGAACCTAATGCCAATGCATATGCTTCCGGCTGGTTTACCAATAATGATGAACATTTAGTGTTTCATTCAGGAACGCTAGATAACTTTTCATCATTTATTTTACTAAATCCAAAACAAAATTATGGAATTGTTGTACTTGCAAATCTAAATTCGGAATATGTACCCAAATTAGTTGAGCATCTTAATACACAAATTGTAAATCACAAGCGATATTCGACGGTTGCGTCTATGCTCAATCAATATAAAGATCAATTTAATATTGTTACCGTTTTGATGACAACACTTATTTTATTAGCATTTATATTCTCAGCTTATCGTGCTTGGCAAATGCGCCATGGTCAAATTCTTTTGCGTAGATCAAAACGGATTGCTGTATTGAGTTGGTTATCATTATGTATATGTATCGCTTTAGCGCTCATATTATATGCATTACCATATCTCATTCTCGGTAGCAATAATTGGTCTTTTGTACTGACTTGGCTACCAATAGAAATTAAATTAGCACTAATCACAACATTAATTGCATTATTCAGTACATTAATTGTAATTCTGTTATTCCTTCATAATGCAGGAACGAAGACATAATAAAAAAGACTTGTTCGAGCCGTGCGTTTGATAATATATCATCCACGATT fmtB amino acidsequence clone: BTEDA22 and BTEDV18MTTKKLYFLSISIIILVAISIAIYTTLNSNTKTRLTNDSQQQIDTIIEHDLQKGHIPGASIL (SEQ IDNO:10) IVKNGKVFLNKGYGYQDVDKKVKASPTTKYEIASNTKAFTGLAILKLAQEGRLNLNDAVSKHVPHFKMNYNGQNETITIKQLLAQTSGIPSDITSEDSVTSKNNRLNDVTHAIMGDELHHKPGEEFEYSNMNYDLLGLIIQNVTKQSYTKYITNSWLKPLHMTHTSFKQTNYKSKHDAIGYELQGSTPVVSKPEFNLWDTPSAYMMTSTEDLEHWIKFQLNPPDKYKSLVQQSHKNLSSTIGEPNANAYASGWFTNNDEHLVFHSGTLDNFSSFILLNPKQNYGIVVLANLNSEYVPKLVEHLNTQIVNHKRYSTVASMLNQYKDQFNIVTVLMTTLILLAFIFSAYRAWQMRHGQILLRRSKRIAVLSWLSLCICIALALILYALPYLILGSNNWSFVLTWLPIEIKLALITTLIALFSTLIVILLFLHTKITKT pbpF nucleotide sequenceclones: BTEBG73 AND BTAJO70CTCTTAAATGAGACCGTTATTTTTTTGTCAAAAAGATAGAAATAATTTCTAAAT (SEQ ID NO:11)TCATATATGATTTAAAGTGAAAGACTTTGAATAGAGGTAGGTAGTTTTGTTAAAAAGACTAAAAGAAAAATCAAATGATGAAATCGTTCAAAATACCATTAACAAGAGAATTAACTTTATATTTGGTGTGATTGTATTTATTTTTGCAGTACTAGTACTACGTTTAGGTTATTTACAAATCGCACAAGGCTCACATTATAAACAAATTATAAAAAATGATGAAAACATTACAGTGAATGAGTCTGTGCCAAGAGGTCGTATTTTAGACAGAAATGGGAAAGTTTTAGTTGATAATGCTTCTAAAATGGCTATTACATATACTAGGGGTCGAAAAACAACACAATCGGAAATGTTGGATACGGCTGAAAAGTTATCAAAGCTAATCAAGATGGATACTAAGAAAATTACAGAACGTGATAAGAAAGATTTCTGGATTCAGTTGCATCCTAAAAAAGCAAAAGCAATGATGACAAAAGAACAAGCTATGTTAGCAGATGGAAGTATTAAACAAGATCAATATGATAAACAACTGTTATCGAAAATCGGAAAATCACAATTAGATGAATTGTCTTCTAAAGATTTACAAGTTTTAGCTATTTTTCGAGAGATGAATGCAGGAACAGTTTTAGATCCACAAATGATAAAAAATGAAGATGTCAGTGAAAAAGAGTATGCAGCAGTTTCTCAGCAACTTTCCAAATTACCAGGTGTTAACACGTCTATGGATTGGGATAGAAAATATCCATATGGCGATACTTTAAGAGGTATATTCGGAGATGTATCGACACCTGCTGAAGGTATTCCAAAAGAATTGACAGAACATTACTTATCCAAAGGATATTCACGCAATGATCGTGTTGGAAAATCTTACCTAGAATATCAATATGAAGATGTATTGCGTGGTAAGAAGAAAGAAATGAAATACACAACGGACAAATCTGGTAAAGTTACATCTTCAGAAGTGTTAAATCCTGGCGCTCGCGGTCAAGATTTGAAATTAACGATCGATATAGATCTTCAAAAAGAAGTAGAAGCATTATTAGATAAACAAATTAAGAAGCTTCGCAGTCAAGGTGCCAAAGATATGGATAATGCAATGATGGTTGTACAAAATCCTAAAAATGGAGACATTCTTGCGCTTGCCGGAAAGCAGATTAATAAGAGTGGTAAAATGACTGATTATGACATTGGTACGTTTACTTCTCAATTTGCGGTTGGATCTTCTGTAAAAGGTGGAACATTATTAGCCGGTTATCAGAATAAAGCTATCAAAGTTGGAGAAACAATGGTCGATGAACCATTACATTTCCAAGGTGGTTTGACAAAACGATCATACTTCAATAAAAACGGGCATGTAACTATTAATGATAAGCAAGCTTTGATGCATTCATCAAACGTATATATGTTTAAAACAGCATTAAAATTAGCGGGAGACCCTTATTATTCTGGTATGGCTTTACCTTCAGACATAAGTTCACCTGCCCAAAAGCTAAGAAGAGGATTAAATCAAGTAGGCTTAGGTGTGAAAACAGGGATAGATTTACCAAATGAAACAAGAGGTCAAATCGAACCATTAACAAATAATCCAGGTAATTATCTAGATTTATCAATTGGTCAATATGATACCTATACACCATTACAATTATCACAATATGTTTCAACTATAGCGAATGATGGTTATAGAATACAGCCACACATTGGATTAACGATTCATGAATCAACTAATAAAGATGAGGTTGGTCCACTCAAGAAGAAAATTAATGGCACTGTCTTGAACAAGGTTAATAATACTGAAAAGGAAATCAAACAAATTCAAGAAGGATTCAAAATGGCATTTAATGATAAAGATGGTACTGGATATGTTAGTTTTAAAGATACAGTAGTACCTACTGCTGGTAAAACGGGTACCGCAGAAGTGTTCCAAAACGGAGAGCCAAGAGTTAACTCTACTTATATAGGATACGCGCCAATTGATGATCCAAAATTAGCGTTTTCAATTGTATATACAAATCAGCCTGTACCACCACCATGGTTAACAGGTGGAGACTTAGGTAGAGATGTAATTAACTACTACTTTAAGCAGTTAGGTAAAGATGATAAAAATAAAGACAAAGACAAATAAAATTTAACCTGACGATTGTGTAGCGCATGGTTGTAAAATTT TAACTTTGC pbpFamino acid sequence clones: BTEBG73 AND BTAJO70LLKRLKEKSNDEIVQNTINKRINFIFGVIVFIFAVLVLRLGYLQIAQGSHYKQIIKNDE (SEQ IDNO:12) NITVNESVPRGRILDRNGKVLVDNASKMAITYTRGRKTTQSEMLDTAEKLSKLIKMDTKKITERDKKDFWIQLHPKKAKAMMTKEQAMLADGSIKQDQYDKQLLSKIGKSQLDELSSKDLQVLAIFREMNAGTVLDPQMIKNEDVSEKEYAAVSQQLSKLPGVNTSMDWDRKYPYGDTLRGIFGDVSTPAEGIPKELTEHYLSKGYSRNDRVGKSYLEYQYEDVLRGKKKEMKYTTDKSGKVTSSEVLNPGARGQDLKLTIDIDLQKEVEALLDKQIKKLRSQGAKDMDNAMMVVQNPKNGDILALAGKQINKSGKMTDYDIGTFTSQFAVGSSVKGGTLLAGYQNKAIKVGETMVDEPLHFQGGLTKRSYFNKNGHVTINDKQALMHSSNVYMFKTALKLAGDPYYSGMALPSDISSPAQKLRRGLNQVGLGVKTGIDLPNETRGQIEPLTNNPGNYLDLSIGQYDTYTPLQLSQYVSTIANDGYRIQPHIGLTIHESTNKDEVGPLKKKINGTVLNKVNNTEKEIKQIQEGFKMAFNDKDGTGYVSFKDTVVPTAGKTGTAEVFQNGEPRVNSTYIGYAPIDDPKLAFSIVYTNQPVPPPWLTGGDLGRDVINYYFKQLGKDDKNKDKDK pbpG nucleotide sequence clones: BTEBU53AND BTEFB55 TCCTATTCCTTATGCATTTCCCCTAATTATAATTAACGTTAAAATAAAAGTCAA (SEQID NO:13) ATTGCCTTAAATATGGTATACTATAACGTAATTTAGGAGGTTAAAGATGACGAATCAAGACAACAATCATCAATTGAATCATCGTATATATCATTTTGAAAAGATATATAAAGCTATCAAACATGTCATTGTTTACATATTTATGATTTTCATTGCCATCGTTGCTATCGCTGTGATTGCGATGTCTTTATATTTTCATCATTTAACTAAAACGTCCGACTCATTATCAGATGATGCTTTAATAAAAAAAGTTCGACAAATACCTGGCGATGAATTATTAGATCATAATAACAAAAATTTATTATATGAGTATAACCATTCTCAAAACTCACTCATTATAGGCCCTAAAACATCAAGTCCAAATGTCATTAAAGCATTAACGTCATCTGAAGACACTTTATTTTATAAACATGATGGCATCTTACCAAAGGCGATTTTAAGAGCAATGATACAAGATATTTTTAATACTGATCAAAGTTCAGGTGGTAGCACAATTACACAACAACTTGTTAAAAATCAAGTTCTTACCAACGAAAAAACATATAGTAGAAAAGCAAATGAACTTCGCCTAGCAATTAGATTAGAACACCTACTCTCAAAAGATGAAATTATATATACATATTTAAATATAGTTCCCTTCGGTAGAGATTATAATGGCGCTAATATTTCCGGAATTGCATCCGCTTCATATAGTCTATTTGGTATTCCACCAAAAGATTTATCAATTGCACAATCTGCATACCTTATCGGTTTGTTGCAAAGCCCATATGGCTATACACCCTACGAAAAAGATGGAACGTTAAAATCGGATAAAGATTTGAAATATAGTATTCAAAGACAACATTATGTATTAAAGCGTATGTTAATCGAAGATCAAATCACTGAAAAAGAATACAACGACGCATTAAAATATGATATTAAATCACATTTGTTAAATCGAAAAAAGCGTTAATTGATGCTCACTTTTTAAAGTAACCACAACAATGAATCCAAATATTAAAA pbpG amino acid sequenceclones: BTEBU53 AND BTEFB55MTNQDNNHQLNHRIYHFEKIYKAIKHVIVYIFMIFIAIVAIAVIAMSLYFHHLTKTSD (SEQ IDNO:14) SLSDDALIKKVRQIPGDELLDHNNKNLLYEYNHSQNSLIIGPKTSSPNVIKALTSSEDTLFYKHDGILPKAILRAMIQDIFNTDQSSGGSTITQQLVKNQVLTNEKTYSRKANELRLAIRLEHLLSKDEIIYTYLNIVPFGRDYNGANISGIASASYSLFGIPPKDLSIAQSAYLIGLLQSPYGYTPYEKDGTLKSDKDLKYSIQRQHYVLKRMLIEDQITEKEYNDALK YDIKSHLLNRKKRcbrA nucleotide sequence clones: BTACA44 and BTAGJ54TAGTCAATGAATAAAGTAATTAAAATGCTTGTTGTTACGCTTGCTTTCCTACTT (SEQ ID NO:15)GTTTTAGCAGGATGTAGTGGGAATTCAAATAAACAATCATCTGATAACAAAGATAAGGAAACAACTTCAATTAAACATGCAATGGGTACAACTGAAATTAAAGGGAAACCAAAGCGTGTTGTTACGCTATATCAAGGTGCCACTGACGTCGCTGTATCTTTAGGTGTTAAACCTGTAGGTGCTGTAGAATCATGGACACAAAAACCGAAATTCGAATACATAAAAAATGATTTAAAAGATACTAAGATTGTAGGTCAAGAACCTGCACCTAACTTAGAGGAAATCTCTAAATTAAAACCGGACTTAATTGTCGCGTCAAAAGTTAGAAATGAAAAAGTTTACGATCAATTATCTAAAATCGCACCAACAGTTTCTACTGATACAGTTTTCAAATTCAAAGATACAACTAAGTTAATGGGGAAAGCTTTAGGGAAAGAAAAAGAAGCTGAAGATTTACTTAAAAAGTACGATGATAAAGTAGCTGCATTCCAAAAAGATGCAAAAGCAAAGTATAAAGATGCATGGCCATTGAAAGCTTCAGTTGTTAACTTCCGTGCTGATCATACAAGAATTTATGCTGGTGGATATGCTGGTGAAATCTTAAATGATTTAGGATTCAAACGTAATAAAGACTTACAAAAACAAGTTGATAATGGTAAAGATATTATCCAACTTACATCTAAAGAAAGCATTCCATTAATGAACGCTGATCATATTTTTGTAGTAAAATCAGATCCAAATGCGAAAGATGCTGCATTAGTTAAAAAGACTGAAAGCGAATGGACTTCAAGTAAAGAGTGGAAAAATTTAGACGCAGTTAAAAACAACCAAGTATCTGATGATTTAGATGAAATCACTTGGAACTTAGCTGGCGGATATAAATCTTCATTAAAACTTATTGACGATTTATATGAAAAGTTAAATATTGAAAAACAATCAAAATAA cbrA amino acidsequence clones: BTACA44 and BTAGJ54MNKVIKMLVVTLAFLLVLAGCSGNSNKQSSDNKDKETTSIKHAMGTTEIKGKPKR (SEQ ID NO:16)VVTLYQGATDVAVSLGVKPVGAVESWTQKPKFEYIKNDLKDTKIVGQEPAPNLEEISKLKPDLIVASKVRNEKVYDQLSKIAPTVSTDTVFKFKDTTKLMGKALGKEKEAEDLLKKYDDKVAAFQKDAKAKYKDAWPLKASVVNFRADHTRIYAGGYAGEILNDLGFKRNKDLQKQVDNGKDIIQLTSKESIPLMNADHIFVVKSDPNAKDAALVKKTESEWTSSKEWKNLDAVKNNQVSDDLDEITWNLAGGYKSSLKLIDDLYEKLNIEKQ SK cbrBnucleotide sequence clones: BTACA44 and BTAGJ54TAATTAAGGAGTTTTACGATGCTACTTAAACCAAAATACCAAATCGTTATTGC (SEQ ID NO:17)TGGTTTATGTCTTGCAATAGTAGCTATCTTAAGTTTAATGATTGGAAATACGCTTGTGTCACCAGGTACGGTGATACAGGCGTTATTCAACTTTGATAGTGAAAACGATTTACATGATGTTGTCACTGGTGCACGGGGCGTCGAGAACAATCATTGCGTTATTGACTGGTGCTGCCCTTGCTGTCTCAGGTTTGTTGATGCAAGCACTTACACGAAACCCAATAGCCTCACCAGGGCTTTTCGGTGTCAATGCAGGCGCAGTATTTTTTGTCATTTTTAGTATTACATTTATCCAAATTCAATCTTTTAAAATGATTGTAGTTATTGCATTTTTGGGGGCTATTGTTGTTACTGTATTAGTTGTTGCACTAGGTATGTTTAGACAAACACTATTCTCACCTCACCGTGTCATTTTGGCAGGTGCTGCGATTGCGATGCTATTTACAGCCTTTACTCAAGGCATACTTATTATGAACGAAACAGACTTACAAGGCCTATTATTTTGGTTAAGTGGCTCCGTTTCATTACGTAATATTTGGGATATCCCATGGATTATTCCGCTTGTATTGATACTTATTTTAATTGCATTTAGCATGGCTGCACACATCAACATCTTGATGACAAGTGACGACATTGCAACCGGCCTCGGTCAAAACATAAAATTAATCAAATGGATGATTATTATGCTCATCAGTATGTTAGCCGGTATTTCGGTAGCCGTAGCTGGATCAATCGTCTTTGTGGGTCTTATCGTACCGAATATTAGCAAACGATTATTACCACCAAACTATAAGTATTTAATTCCTTTTACTGCATTAGCTGGAGCAATCCTAATGATCATTTCAGACATTGTTGCTCGTATAATAATTAAGCCACTAGAGTTGCCTATCGGTGTCGTTACCGCTGTCATTGGCGCTATTGTCTTAATCTATATTATGAAGAAAGGACGTCAACGCTTATGA cbrB amino acidsequences clones: BTACA44 and BTAGJ54MLLKPYQIVIAGLCLAIVAILSLMIGNTLVSPGTVIQALFNFDSENDLHDVVTGAR (SEQ ID NO:18)ASRTIIALLTGAALAVSGLLMQALTRNPIASPGLFGVNAGAVFFVIFSITFIQIQSFKMIVVIAFLGAIVVTVLVVALGMFRQTLFSPHRVILAGAAIAMLFTAFTQGILIMNETDLQGLLFWLSGSVSLRNIWDIPWIIPLVILILIAFSMAAHINILMTSDDIATGLGQNIKLIKWMIIMLISMLAGISVAVAGSIVFVGLIVPNISKRLLPPNYKYLIPFTALAGAILMIISDIVARIIIKPLELPIGVVTAVIGAIVLIYIMKKGRQRL cbrC nucleotide sequence clones:BTACA44 and BTAGJ54TAAGCCACTAGAGTTGCCTATCGGTGTCGTTACCGCTGTCATTGGCGCTATTGT (SEQ ID NO:19)CTTAATCTATATTATGAAGAAAGGACGTCAACGCTTATGACCGAAAAGATTAATAAAAAAGACAATTACCATCTCATCTTCGCGTTAATCTTTTTAGCCATCGTTTCAGTGGTAAGTATGATGATTGGTTCAAGCTTTATACCATTACAACGCGTACTGATGTACTTTATAAATCCAAATGACAGTATGGATCAATTCACTTTAGAAGTATTACGCTTACCTCGCATTACACTTGCGATTTTAGCAGGTGCCGCACTAGGAATGAGTGGTTTAATGTTGCAAAATGTATTAAAAAATCCAATTGCCTCACCTGATATTATCGGTATCACAGGTGGTGCTAGCTTAAGTGCTGTTGTCTTTATTGCATTTTTCAGCCATTTAACAATACATTTACTTCCACTATTTGCAGTATTAGGTGGCGCAGTTGCAATGATGATACTATTAGTGTTTCAAACGAAAGGACAAATACGCCCGACAACACTCATAATCATCGGTATTTCGATGCAAACGTTGTTTATTGCGCTTGTCCAAGGATTACTCATTACAACGAAGCAATTATCTGCTGCCAAAGCTTATACATGGCTAGTCGGAAGTCTTTACGGTGCTACGTTTAAAGATACAATCATTTTGGGTATGGTTATTTTAGCTGTTGTGCCGTTGTTATTTCTTGTTATACCAAAAATGAAAATATCTATACTTGATGACCCTGTAGCGATTGGCTTAGGCTTACATGTACAACGTATGAAACTAATCCAATTAATCACTTCTACTATACTCGTATCTATGGCAATCAGTTTAGTAGGTAACATTGGGTTTGTCGGTTTAATCGCACCACATATCGCGAAAACAATCGTTCGCGGAAGTTATGCTAAAAAGTTACTAATGTCAGCAATGATTGGTGCCATATCAATTGTTATTGCAGACTTAATTGGGCGTACCTTATTCTTGCCTAAAGAAGTGCCAGCAGGTGTATTTATTGCTGCTTTTGGTGCCCCATTCTTCATATACTTATTATTAACCGTGAAAAAGTTATAA cbrC amino acid sequences clones: BTACA44 and BTAGJ54MTEKINKKDNYHLIFALIFLAIVSVVSMMIGSSFIPLQRVLMYFINPNDSMDQFTLE (SEQ ID NO:20)VLRLPRITLAILAGAALGMSGLMLQNVLKNPIASPDIIGITGGASLSAVVFIAFFSHLTIHLLPLFAVLGGAVAMMILLVFQTKGQIRPTTLIIIGISMQTLFIALVQGLLITTKQLSAAKAYTWLVGSLYGATFKDTIILGMVILAVVPLLFLVIIPRMKISILDDPVAIGLGLHVQRMKLIQLITSTILVSMAISLVGNIGFVGLIAPHIAKTIVRGSYAKKLLMSAMIGAISIVIADLIGRTLFLPKEVPAGVFIAAFGAPFFIYLLLTVKKL enolase nucleotide sequenceclones: BTAAI44 and BTAGE12TAATGACACTTATTTTTTGAAAATAATAGTAATATCATTTTGTTAAATGAAAGA (SEQ ID NO:21)ATAAAGCTATAATAATTATAGAATAACTATTTAAAGGAGATTATAAACATGCCAATTATTACAGATGTTTACGCTCGCGAAGTCTTAGACTCTCGTGGTAACCCAACTGTTGAAGTAGAAGTATTAACTGAAAGTGGCGCATTTGGTCGTGCATTAGTACCATCAGGTGCTTCAACTGGTGAACACGAAGCTGTTGAATTACGTGATGGAGACAAATCACGTTATTTAGGTAAAGGTGTTACTAAAGCAGTTGAAAACGTTAATGAAATCATCGCACCAGAAATTATTGAAGGTGAATTTTCAGTATTAGATCAAGTATCTATTGATAAAATGATGATCGCATTAGACGGTACTCCAAACAAAGGTAAATTAGGTGCAAATGCTATTTTAGGTGTATCTATCGCAGTAGCACGTGCAGCAGCTGACTTATTAGGTCAACCACTTTACAAATATTTAGGTGGATTTAATGGTAAGCAGTTACCAGTACCAATGATGAACATCGTTAATGGTGGTTCTCACTCAGATGCTCCAATTGCATTCCAAGAATTCATGATTTTACCTGTAGGTGCTACAACGTTCAAAGAATCATTACGTTGGGGTACTGAAATTTTCCACAACTTAAAATCAATTTTAAGCCAACGTGGTTTAGAAACTGCCGTAGGTGACGAAGGTGGTTTCGCTCCTAAATTTGAAGGTACTGAAGATGCTGTTGAAACAATTATCCAAGCAATCGAAGCAGCTGGTTACAAACCAGGTGAAGAAGTATTCTTAGGATTTGACTGTGCATCATCAGAATTCTATGAAAATGGTGTATATGACTACAGTAAGTTCGAAGGCGAACACGGTGCAAAACGTACAGCTGCAGAACAAGTTGACTACTTAGAACAATTAGTAGACAAATATCCTATCATTACAATTGAAGACGGTATGGACGAAAACGACTGGGATGGTTGGAAACAACTTACAGAACGTATCGGTGACCGTGTACAATTAGTAGGTGACGATTTATTCGTAACAAACACTGAAATTTTAGCAAAAGGTATTGAAAACGGAATTGGTAACTCAATCTTAATTAAAGTTAACCAAATCGGTACATTAACTGAAACATTTGATGCAATCGAAATGGCTCAAAAAGCTGGTTACACAGCAGTAGTTTCTCACCGTTCAGGTGAAACAGAAGATACAACAATTGCTGATATTGCTGTTGCTACAAACGCTGGTCAAATTAAAACTGGTTCATTATCACGTACTGACCGTATTGCTAAATACAATCAATTATTACGTATCGAAGATGAATTATTTGAAACTGCTAAATATGACGGTATCAAATCATTCTATAACTTAGATAAATAATTTTCTTTATAATCAAATGCTGACATAATTTTAGTTGAGGATTATTATGACGG enolase amino acid sequence clones:BTAAI44 and BTAGE12MPIITDVYAREVLDSRGNPTVEVEVLTESGAFGRALVPSGASTGEHEAVELRDGD (SEQ ID NO:22)KSRYLGKGVTKAVENVNEIIAPEIIEGEFSVLDQVSIDKMMIALDGTPNKGKLGANAILGVSIAVARAAADLLGQPLYKYLGGFNGKQLPVPMMNIVNGGSHSDAPIAFQEFMILPVGATTFKESLRWGTEIFHNLKSIISQRGLETAVGDEGGFAPKFEGTEDAVETIIQAIEAAGYKPGEEVFLGFDCASSEFYENGVYDYSKFEGEHGAKRTAAEQVDYLEQLVDKYPIITIEDGMDENDWDGWKQLTERIGDRVQLVGDDLFVTNTEILAKGIENGIGNSILIKVNQIGTLTETFDAIEMAQKAGYTAVVSHRSGETEDTTIADIAVATNAGQIKTGSLSRTDRIAKYNQLLRIEDELFETAKYDGIKSFYNLDK

Illustrative of the invention, the nucleic acid molecule described inTable 1 was discovered in a DNA library derived from a S. aureus ISP3genomic DNA.

Nucleic acid molecules of the present invention may be in the form ofRNA, such as mRNA, or in the form of DNA, including, for instance, DNAand genomic DNA obtained by cloning or produced synthetically. The DNAmay be double-stranded or single-stranded. Single-stranded DNA or RNAmay be the coding strand, also known as the sense strand, or it may bethe non-coding strand, also referred to as the anti-sense strand.

By “isolated” polynucleotide sequence is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. This includes segments of DNA comprising the S. aureuspolynucleotides of the present invention isolated from the nativechromosome. These fragments include both isolated fragments consistingonly of S. aureus DNA and fragments comprising heterologous sequencessuch as vector sequences or other foreign DNA. For example, recombinantDNA molecules contained in a vector are considered isolated for thepurposes of the present invention which may be partially orsubstantially purified. Further examples of isolated DNA moleculesinclude recombinant DNA molecules introduced and maintained inheterologous host cells or purified (partially or substantially) DNAmolecules in solution. Isolated RNA molecules include in vivo or invitro RNA transcripts of the DNA molecules of the present invention.Isolated nucleic acid molecules according to the present inventionfurther include such molecules produced synthetically which may bepartially or substantially purified. The term “isolated” does not referto genomic or cDNA libraries, whole cell mRNA preparations, genomic DNAdigests (including those gel separated by electrophoresis), shearedwhole cell genomic DNA preparations or other compositions where the artdemonstrates no distinguishing features of the polynucleotides sequencesof the present invention.

In addition, isolated nucleic acid molecules of the invention includeDNA molecules which comprise a sequence substantially different fromthose described above but which, due to the degeneracy of the geneticcode, still encode a S. aureus polypeptides and peptides of the presentinvention (e.g. polypeptides of Table 1). That is, all possible DNAsequences that encode the S. aureus polypeptides of the presentinvention. This includes the genetic code and species-specific codonpreferences known in the art. Thus, it would be routine for one skilledin the art to generate the degenerate variants described above, forinstance, to optimize codon expression for a particular host (e.g.,change codons in the bacteria mRNA to those preferred by a mammalian orother bacterial host such as E. coli).

The invention further provides isolated nucleic acid molecules havingthe nucleotide sequence shown in Table 1 or a nucleic acid moleculehaving a sequence complementary to one of the above sequences. Suchisolated molecules, particularly DNA molecules, are useful as probes forgene mapping and for identifying S. aureus in a biological sample, forinstance, by PCR or Northern blot analysis. In specific embodiments, thepolynucleotides of the present invention are less than 300 kb, 200 kb,100 kb, 50 kb, 10,kb, 7.5 kb, 5 kb, 2.5 kb, and 1 kb. In anotherembodiment, the polynucleotides comprising the coding sequence forpolypeptides of the present

The present invention is further directed to nucleic acid moleculesencoding portions or fragments of the polynucleotide sequences describedherein, e.g., shown in the Tables, sequence listing, or contained in thedeposited clones. Uses for the polynucleotide fragments of the presentinvention include probes, primers, molecular weight, markers and forexpressing the polypeptide fragments of the present invention. Fragmentsinclude portions of the polynucleotide sequences, at least 10 contiguousnucleotides in length selected from any two integers, one of whichrepresenting a 5′ nucleotide position and a second of which representinga 3′ nucleotide position, where the first, or 5′ most, nucleotide foreach disclosed polynucleotide sequence is position 1. That is, everycombination of a 5′ and 3′ nucleotide position that a fragment at least10 contiguous nucleotides in length could occupy is included in theinvention as an individual specie. “At least” means a fragment may be 10contiguous nucleotide bases in length or any integer between 10 and thelength of an entire nucleotide sequence minus 1. Therefore, included inthe invention are contiguous fragments specified by any 5′ and 3′nucleotide base positions of a polynucleotide sequences wherein thecontiguous fragment is any integer between 10 and the length of anentire nucleotide sequence minus 1. The polynucleotide fragmentsspecified by 5′ and 3′ positions can be immediately envisaged using theabove description and are therefore not individually listed solely forthe purpose of not unnecessarily lengthening the specifications.Although it is particularly pointed out that each of the above describedspecies are included in the present invention.

Further, the invention includes polynucleotides comprising sub-genusesof fragments specified by size, in nucleotides, rather than bynucleotide positions. The invention includes any fragment size, incontiguous nucleotides, selected from integers between 10 and the lengthof an entire nucleotide sequence minus 1 (where 1 is the first, or 5′most, nucleotide for each disclosed polynucleotide sequence). Preferredsizes of contiguous nucleotide fragments include 20 nucleotides, 30nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 125nucleotides, 150 nucleotides, 175 nucleotides, 200 nucleotides, 250nucleotides, 300 nucleotides, 350 nucleotides, 400 nucleotides, 450nucleotides, 500 nucleotides, 550 nucleotides, 600 nucleotides, 650nucleotides, 700 nucleotides, 750 nucleotides, 800 nucleotides, 850nucleotides, 900 nucleotides, 950 nucleotides, 1000 nucleotides. Otherpreferred sizes of contiguous polynucleotide fragments, which may beuseful as diagnostic probes and primers, include fragments 50-300nucleotides in length which include, as discussed above, fragment sizesrepresenting each integer between 50-300. Larger fragments are alsouseful according to the present invention corresponding to most, if notall, of the polynucleotide sequences of the sequence listing ordeposited clones. The preferred sizes are, of course, meant to exemplifynot limit the present invention as all size fragments, representing anyinteger between 10 and the length of an entire nucleotide sequence minus1 of the sequence listing or deposited clones, are included in theinvention. Additional preferred nucleic acid fragments of the presentinvention include nucleic acid molecules encoding epitope-bearingportions of the polypeptides.

The polynucleotide fragments, specified in contiguous nucleotides, canbe immediately envisaged using the above description and are thereforenot individually listed solely for the purpose of not unnecessarilylengthening the specification.

The present invention also provides for the exclusion of any fragment,specified by 5′ and 3′ base positions or by size in nucleotide bases asdescribed above for any nucleotide sequence of the sequence listing ordeposited clones. Any number of fragments of nucleotide sequencesspecified by 5′ and 3′ base positions or by size in nucleotides, asdescribed above, may be excluded from the present invention.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of a polynucleotide in a nucleicacid molecules of the invention described above, for instance,nucleotide sequences of Table 1 or the S. aureus sequences of theplasmid clones listed in Table 1. By “stringent hybridizationconditions” is intended overnight incubation at 42° C. in a solutioncomprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65° C.

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides bases, and more preferably at least about 20nucleotides bases, still more preferably at least about 30 nucleotidesbases, and even more preferably about 30-70 (e.g., 50) nucleotides basesof the reference polynucleotide. These are useful as diagnostic probesand primers as discussed above. By a portion of a polynucleotide of “atleast 20 nucleotides bases in length,” for example, is intended 20 ormore contiguous nucleotides bases nucleotides from the nucleotidesequence of the reference polynucleotide (e.g., the nucleotide sequenceas shown in Table 1). Portions of a polynucleotide which hybridizes to anucleotide sequence in Table 1, which can be used as probes and primers,may also be precisely specified by 5′ and 3′ base positions or by sizein nucleotide bases as described above or precisely excluded in the samemanner.

The nucleic acid molecules of the present invention, which encode a S.aureus polypeptide, may include, but are not limited to, nucleic acidmolecules encoding: the full length S. aureus polypeptide of Table 1,the full length polypeptide expressed by the plasmid clones listed inTable 1, and portions of the S aureus polypeptides of Table 1 and thepolypeptides expressed by the plasmid clones listed in Table 1. Alsoincluded in the present invention are nucleic acids encoding the abovefull length sequences and further comprise additional sequences, such asthose encoding an added secretory leader sequence, such as a pre-, orpro- or prepro-protein sequence. Further included in the presentinvention are nucleic acids encoding the above full length sequences andportions thereof and further comprise additional heterologous amino acidsequences encoded by nucleic acid sequences from a different source.

Also included in the present invention are nucleic acids encoding theabove protein sequences together with additional, non-coding sequences,including for example, but not limited to non-coding 5′ and 3′sequences. These sequences include transcribed, non-translated sequencesthat may play a role in transcription, and mRNA processing, for example,ribosome binding and stability of mRNA. Also included in the presentinvention are additional coding sequences which provide additionalfunctionalities.

Thus, a nucleotide sequence encoding a polypeptide may be fused to amarker sequence, such as a sequence encoding a peptide which facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. For instance,hexa-histidine provides for convenient purification of the fusionprotein. See Gentz et al. (1989) Proc. Natl. Acad. Sci. 86:821-24. The“HA” tag is another peptide useful for purification which corresponds toan epitope derived from the influenza hemagglutinin protein. See Wilsonet al. (1984) Cell 37:767. As discussed below, other such fusionproteins include the S. aureus fused to Fc at the N- or C-terminus.

Variant and Mutant Polynucleotides

The present invention further relates to variants of the nucleic acidmolecules which encode portions, analogs or derivatives of a S. aureuspolypeptides of Table 1, or encoded by the plasmid clones listed inTable 1, and variant polypeptides thereof including portions, analogs,and derivatives of the S. aureus polypeptides. Variants may occurnaturally, such as a natural allelic variant. By an “allelic variant” isintended one of several alternate forms of a gene occupying a givenlocus on a chromosome of an organism. See, e.g., B. Lewin, Genes IV(1990). Non-naturally occurring variants may be produced using art-knownmutagenesis techniques.

Such nucleic acid variants include those produced by nucleotidesubstitutions, deletions, or additions. The substitutions, deletions, oradditions may involve one or more nucleotides. The variants may bealtered in coding regions, non-coding regions, or both. Alterations inthe coding regions may produce conservative or non-conservative aminoacid substitutions, deletions or additions. Especially preferred amongthese are silent substitutions, additions and deletions, which do notalter the properties and activities of a S. aureus protein of thepresent invention or portions thereof. Also especially preferred in thisregard are conservative substitutions.

Such polypeptide variants include those produced by amino acidsubstitutions, deletions or additions. The substitutions, deletions, oradditions may involve one or more residues. Alterations may produceconservative or non-conservative amino acid substitutions, deletions, oradditions. Especially preferred among these are silent substitutions,additions and deletions, which do not alter the properties andactivities of a S. aureus protein of the present invention or portionsthereof. Also especially preferred in this regard are conservativesubstitutions.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofS. aureus polypeptides or peptides by recombinant techniques.

The present application is directed to nucleic acid molecules at least90%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequenceshown in Table 1 or to the nucleic acid sequence of the plasmid cloneslisted in Table 1. The above nucleic acid sequences are includedirrespective of whether they encode a polypeptide having S. aureusactivity. This is because even where a particular nucleic acid moleculedoes not encode a polypeptide having S. aureus activity, one of skill inthe art would still know how to use the nucleic acid molecule, forinstance, as a hybridization probe. Uses of the nucleic acid moleculesof the present invention that do not encode a polypeptide having S.aureus activity include, inter alia, isolating an S. aureus gene orallelic variants thereof from a DNA library, and detecting S. aureusmRNA expression samples, environmental samples, suspected of containingS. aureus by Northern Blot analysis.

Preferred, are nucleic acid molecules having sequences at least 90%,95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shownin Table 1 or to the nucleic acid sequence of the plasmid clones listedin Table 1, which do, in fact, encode a polypeptide having S. aureusprotein activity By “a polypeptide having S. aureus activity” isintended polypeptides exhibiting activity similar, but not necessarilyidentical, to an activity of the S. aureus protein of the invention, asmeasured in a particular biological assay suitable for measuringactivity of the specified protein.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequence of the plasmid cloneslisted in Table 1 or a nucleic acid sequence shown in Table 1 willencode a polypeptide having S. aureus protein activity. In fact, sincedegenerate variants of these nucleotide sequences all encode the samepolypeptide, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving S. aureus protein activity. This is because the skilled artisanis fully aware of amino acid substitutions that are either less likelyor not likely to significantly effect protein function (e.g., replacingone aliphatic amino acid with a second aliphatic amino acid), as furtherdescribed below.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the S.aureus polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted, inserted, or substituted with another nucleotide. The querysequence may be an entire sequence shown in Table 1, the ORF (openreading frame), or any fragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to anucleotide sequence of the presence invention can be determinedconventionally using known computer programs. A preferred method fordetermining the best overall match between a query sequence (a sequenceof the present invention) and a subject sequence, also referred to as aglobal sequence alignment, can be determined using the FASTDB computerprogram based on the algorithm of Brutlag et al. See Brutlag et al.(1990) Comp. App. Biosci. 6:237-245. In a sequence alignment the queryand subject sequences are both DNA sequences. An RNA sequence can becompared by first converting U's to T's. The result of said globalsequence alignment is in percent identity. Preferred parameters used ina FASTDB alignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only nucleotidesoutside the 5′ and 3′ nucleotides of the subject sequence, as displayedby the FASTDB alignment, which are not matched/aligned with the querysequence, are calculated for the purposes of manually adjusting thepercent identity score.

For example, a 90 nucleotide subject sequence is aligned to a 100nucleotide query sequence to determine percent identity. The deletionsoccur at the 5′ end of the subject sequence and therefore, the FASTDBalignment does not show a matched/alignment of the first 10 nucleotidesat 5′ end. The 10 unpaired nucleotides represent 10% of the sequence(number of nucleotides at the 5′ and 3′ ends not matched/total number ofnucleotides in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90nucleotides were perfectly matched the final percent identity would be90%. In another example, a 90 nucleotide subject sequence is comparedwith a 100 nucleotide query sequence. This time the deletions areinternal deletions so that there are no nucleotides on the 5′ or 3′ ofthe subject sequence which are not matched/aligned with the query. Inthis case the percent identity calculated by FASTDB is not manuallycorrected. Once again, only nucleotides 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

Vectors and Host Cell

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells comprising therecombinant vectors, and the production of S. aureus polypeptides andpeptides of the present invention expressed by the host cells.

Recombinant constructs may be introduced into host cells using wellknown techniques such as infection, transduction, transfection,transvection, electroporation and transformation. The vector may be, forexample, a phage, plasmid, viral or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

Preferred are vectors comprising cis-acting control regions to thepolynucleotide of interest. Appropriate trans-acting factors may besupplied by the host, supplied by a complementing vector or supplied bythe vector itself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression, which may be inducible and/or cell type-specific.Particularly preferred among such vectors are those inducible byenvironmental factors that are easy to manipulate, such as temperatureand nutrient additives.

Expression vectors useful in the present invention include chromosomal-,episomal- and virus-derived vectors, e.g., vectors derived frombacterial plasmids, bacteriophage, yeast episomes, yeast chromosomalelements, viruses such as baculoviruses, papova viruses, vacciniaviruses, adenoviruses, fowl pox viruses, pseudorabies viruses andretroviruses, and vectors derived from combinations thereof, such ascosmids and phagemids.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled artisan. The expression constructs will further contain sitesfor transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will preferablyinclude a translation initiating site at the beginning and a terminationcodon (UAA, UGA or UAG) appropriately positioned at the end of thepolypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture and tetracycline,kanamycin, or ampicillin resistance genes for culturing in E. coli andother bacteria. Representative examples of appropriate hosts include,but are not limited to, bacterial cells, such as E. coli, Streptomycesand Salmonella typhimurium cells; fungal cells, such as yeast cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS and Bowes melanoma cells; and plant cells.Appropriate culture mediums and conditions for the above-described hostcells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE9, pQE10 available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pN16a, pNH18A, pNH46A available fromStratagene; pET series of vectors available from Novagen; and ptrc99a,pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Amongpreferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSGavailable from Stratagene; and pSVK3, pBPV, pMSG and pSVL available fromPharmacia. Other suitable vectors will be readily apparent to theskilled artisan.

Among known bacterial promoters suitable for use in the presentinvention include the E. coli lacI and lacZ promoters, the T3, T5 and T7promoters, the gpt promoter, the lambda PR and PL promoters and the trppromoter. Suitable eukaryotic promoters include the CMV immediate earlypromoter, the HSV thymidine kinase promoter, the early and late SV40promoters, the promoters of retroviral LTRs, such as those of the Roussarcoma virus (RSV), and metallothionein promoters, such as the mousemetallothionein-I promoter.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals (for example, Davis, et al., Basic Methods InMolecular Biology (1986)).

Transcription of DNA encoding the polypeptides of the present inventionby higher eukaryotes may be increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 nucleotides that act to increase transcriptional activityof a promoter in a given host cell-type. Examples of enhancers includethe SV40 enhancer, which is located on the late side of the replicationorigin at nucleotides 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

For secretion of the translated polypeptide into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide, for example, the amino acidsequence KDEL. The signals may be endogenous to the polypeptide or theymay be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, may be added to theN-terminus of the polypeptide to improve stability and persistence inthe host cell, during purification, or during subsequent handling andstorage. Also, peptide moieties may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizeproteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A 0232 262). On theother hand, for some uses it would be desirable to be able to delete theFc part after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when Feportion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as antigen forimmunizations. In drug discovery, for example, human proteins, such as,hIL5-receptor has been fused with Fe portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. SeeBennett, D. et al. (1995) J. Molec. Recogn. 8:52-58 and Johanson, K. etal. (1995) J. Biol. Chem. 270 (16):9459-9471.

The S. aureus polypeptides can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, lectin chromatography and high performance liquidchromatography (“HPLC”) is employed for purification. Polypeptides ofthe present invention include naturally purified products, products ofchemical synthetic procedures, and products produced by recombinanttechniques from a prokaryotic or eukaryotic host, including, forexample, bacterial, yeast, higher plant, insect and mammalian cells.

Polypeptides and Fragments

The invention further provides an isolated S. aureus polypeptide havingthe amino acid sequence encoded by a plasmid clone listed in Table 1, oran amino acid sequence in Table 1, or a peptide or polypeptidecomprising a portion of the above polypeptides.

Variant and Mutant Polypeptides

To improve or alter the characteristics of S. aureus polypeptides of thepresent invention, protein engineering may be employed. Recombinant DNAtechnology known to those skilled in the art can be used to create novelmutant proteins or muteins including single or multiple amino acidsubstitutions, deletions, additions, or fusion proteins. Such modifiedpolypeptides can show, e.g., enhanced activity or increased stability.In addition, they may be purified in higher yields and show bettersolubility than the corresponding natural polypeptide, at least undercertain purification and storage conditions.

N-Terminal and C-Terminal Deletion Mutants

It is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus without substantial loss of biologicalfunction. For instance, Ron et al. J. Biol. Chem., 268:2984-2988 (1993),reported modified KGF proteins that had heparin binding activity even if3, 8, or 27 N-terminal amino acid residues were missing. Accordingly,the present invention provides polypeptides having one or more residuesdeleted from the amino terminus of the amino acid sequence of the S.aureus polypeptides shown in Table 1, and polynucleotides encoding suchpolypeptides.

Similarly, many examples of biologically functional C-terminal deletionmuteins are known. For instance, Interferon gamma shows up to ten timeshigher activities by deleting 8-10 amino acid residues from the carboxyterminus of the protein See, e.g., Dobeli, et al. (1988) J.Biotechnology 7:199-216. Accordingly, the present invention providespolypeptides having one or more residues from the carboxy terminus ofthe amino acid sequence of the S. aureus polypeptides shown in Table 1.The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini as described below.

The present invention is further directed to polynucleotide encodingportions or fragments of the amino acid sequences described herein aswell as to portions or fragments of the isolated amino acid sequencesdescribed herein. Fragments include portions of the amino acid sequencesof the Tables, sequence listing, and encoded by deposited cDNA clones,at least 7 contiguous amino acid in length, selected from any twointegers, one of which representing a N-terminal position and anotherrepresenting a C-terminal position. The first, or N-terminal most, codonof each polypeptide disclosed herein is position 1. Every combination ofa N-terminal and C-terminal position that a fragment at least 7contiguous amino acid residues in length could occupy, on any givenamino acid sequence is included in the invention as an individualspecie. At least means a fragment may be 7 contiguous amino acidresidues in length or any integer between 7 and the number of residuesin a full length amino acid sequence minus 1. Therefore, included in theinvention are species of contiguous fragments specified by anyN-terminal and C-terminal positions of amino acid sequence set forth inthe sequence listing or encoded by the deposited cDNA clones, whereinthe contiguous fragment is any integer between 7 and the number ofresidues in a full length sequence minus 1. The polypeptide fragmentsspecified by N-terminal and C-terminal positions can be immediatelyenvisaged using the above description and are therefore not individuallylisted solely for the purpose of not unnecessarily lengthening thespecification. Although it is particularly pointed out that each of theabove described species are included in the present invention.

Further, the invention includes polypeptides comprising sub-genuses offragments specified by size, in amino acid residues, rather than byN-terminal and C-terminal positions. The invention includes any fragmentsize, in contiguous amino acid residues, selected from integers between7 and the number of residues in a full length sequence minus 1.Preferred sizes of contiguous polypeptide fragments include at least 7amino acid residues, at least 10 amino acid residues, at least 20 aminoacid residues, at least 30 amino acid residues, at least 40 amino acidresidues, at least 50 amino acid residues, at least 75 amino acidresidues, at least 100 amino acid residues, at least 125 amino acidresidues, at least 150 amino acid residues, at least 175 amino acidresidues, at least 200 amino acid residues, at least 225 amino acidresidues, at least 250 amino acid residues, at least 275 amino acidresidues, at least 300 amino acid residues, at least 325 amino acidresidues, at least 350 amino acid residues, at least 375 amino acidresidues, at least 400 amino acid residues, at least 425 amino acidresidues, and at least 450 amino acid residues. The preferred sizes are,of course, meant to exemplify, not limit, the present invention as allsize fragments representing any integer between 7 and the number ofresidues in a full length sequence minus 1 are included in theinvention.

The contiguous polypeptide fragments specified by size in amino acidresidues of the present invention can be immediately envisaged using theabove description and are therefore not individually listed solely forthe purpose of not unnecessarily lengthening the specification.

The present invention also provides for the exclusion of any fragmentsspecified by N-terminal and C-terminal positions or by size in aminoacid residues as described above. Any number of fragments specified byN-terminal and C-terminal positions or by size in amino acid residues asdescribed above.

It is particularly pointed out that the above fragments need not beactive since they would be useful, for example, in immunoassays, inepitope mapping, epitope tagging, to generate antibodies to a particularportion of the polypeptide, as vaccines, and as molecular weightmarkers.

Also preferred are polypeptide and polynucleotide fragmentscharacterized by structural or functional domains, such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

Other preferred fragments are biologically active fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the polypeptide of thepresent invention. The biological activity of the fragments may includean improved desired activity, or a decreased undesirable activity.

Other Mutants

In addition to N- and C-terminal deletion forms of the protein discussedabove, it also will be recognized by one of ordinary skill in the artthat some amino acid sequences of the S. aureus polypeptide can bevaried without significant effect of the structure or function of theprotein. If such differences in sequence are contemplated, it should beremembered that there will be critical areas on the protein whichdetermine activity.

Thus, the invention further includes variations of the S. aureuspolypeptides which show substantial S. aureus polypeptide activity orwhich include regions of S. aureus protein such as the protein portionsdiscussed below. Such mutants include deletions, insertions, inversions,repeats, and type substitutions selected according to general rulesknown in the art so as to have little effect on activity. For example,guidance concerning how to make phenotypically silent amino acidsubstitutions is provided. There are two main approaches for studyingthe tolerance of an amino acid sequence to change. See, Bowie, J. U. etal. (1990), Science 247:1306-1310. The first method relies on theprocess of evolution, in which mutations are either accepted or rejectedby natural selection. The second approach uses genetic engineering tointroduce amino acid changes at specific positions of a cloned gene andselections or screens to identify sequences that maintain functionality.

These studies have revealed that proteins are surprisingly tolerant ofamino acid substitutions. The studies indicate which amino acid changesare likely to be permissive at a certain position of the protein. Forexample, most buried amino acid residues require nonpolar side chains,whereas few features of surface side chains are generally conserved.Other such phenotypically silent substitutions are described by Bowie etal. (supra) and the references cited therein. Typically seen asconservative substitutions are the replacements, one for another, amongthe aliphatic amino acids Ala, Val, Leu and Ile; interchange of thehydroxyl residues Ser and Thr, exchange of the acidic residues Asp andGlu, substitution between the amide residues Asn and Gln, exchange ofthe basic residues Lys and Arg and replacements among the aromaticresidues Phe, Tyr.

Thus, the fragment, derivative, analog, or homolog of the polypeptide ofTable 1, or that encoded by the plasmids listed in Table 1, may be: (i)one in which one or more of the amino acid residues are substituted witha conserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code: or (ii) one in which one or moreof the amino acid residues includes a substituent group: or (iii) one inwhich the S. aureus polypeptide is fused with another compound, such asa compound to increase the half-life of the polypeptide (for example,polyethylene glycol): or (iv) one in which the additional amino acidsare fused to the above form of the polypeptide, such as an IgG Fc fusionregion peptide or leader or secretory sequence or a sequence which isemployed for purification of the above form of the polypeptide or aproprotein sequence. Such fragments, derivatives and analogs are deemedto be within the scope of those skilled in the art from the teachingsherein.

Thus, the S. aureus polypeptides of the present invention may includeone or more amino acid substitutions, deletions, or additions, eitherfrom natural mutations or human manipulation. As indicated, changes arepreferably of a minor nature, such as conservative amino acidsubstitutions that do not significantly affect the folding or activityof the protein (see Table 3).

TABLE 3 Conservative Amino Acid Substitutions. Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

Amino acids in the S. aureus proteins of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis. See,e.g., Cunningham et al. (1989) Science 244:1081-1085. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity using assays appropriate for measuring the function of theparticular protein.

Of special interest are substitutions of charged amino acids with othercharged or neutral amino acids which may produce proteins with highlydesirable improved characteristics, such as less aggregation.Aggregation may not only reduce activity but also be problematic whenpreparing pharmaceutical formulations, because aggregates can beimmunogenic. See, e.g., Pinckard et al., (1967) Clin. Exp. Immunol.2:331-340; Robbins, et al., (1987) Diabetes 36:838-845; Cleland, et al.,(1993) Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of the S. aureus polypeptide can besubstantially purified by the one-step method described by Smith et al.(1988) Gene 67:31-40. Polypeptides of the invention also can be purifiedfrom natural or recombinant sources using antibodies directed againstthe polypeptides of the invention in methods which are well known in theart of protein purification.

The invention further provides for isolated S. aureus polypeptidescomprising an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of a full-length S. aureus polypeptidehaving the complete amino acid sequence shown in Table 1; (b) the aminoacid sequence of a full-length S. aureus polypeptide having the completeamino acid sequence shown in Table 1 excepting the N-terminalmethionine; (c) the complete amino acid sequence encoded by the plasmidslisted in Table 1; and (d) the complete amino acid sequence exceptingthe N-terminal methionine encoded by the plasmids listed in Table 1. Thepolypeptides of the present invention also include polypeptides havingan amino acid sequence at least 80% identical, more preferably at least90% identical, and still more preferably 95%, 96%, 97%, 98% or 99%identical to those described in (a), (b), (c), and (d) above.

Further polypeptides of the present invention include polypeptides whichhave at least 90% similarity, more preferably at least 95% similarity,and still more preferably at least 96%, 97%, 98% or 99% similarity tothose described above.

A further embodiment of the invention relates to a polypeptide whichcomprises the amino acid sequence of a S. aureus polypeptide having anamino acid sequence which contains at least one conservative amino acidsubstitution, but not more than 50 conservative amino acidsubstitutions, not more than 40 conservative amino acid substitutions,not more than 30 conservative amino acid substitutions, and not morethan 20 conservative amino acid substitutions. Also provided arepolypeptides which comprise the amino acid sequence of a S. aureuspolypeptide, having at least one, but not more than 10, 9, 8, 7, 6, 5,4, 3, 2 or 1 conservative amino acid substitutions.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, (indels) or substituted withanother amino acid. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequences shown in Table 1 or to the amino acid sequence encoded bythe plasmids listed in Table 1 can be determined conventionally usingknown computer programs. A preferred method for determining the bestoverall match between a query sequence (a sequence of the presentinvention) and a subject sequence, also referred to as a global sequencealignment, can be determined using the FASTDB computer program based onthe algorithm of Brutlag et al., (1990) Comp. App. Biosci. 6:237-245. Ina sequence alignment the query and subject sequences are both amino acidsequences. The result of said global sequence alignment is in percentidentity. Preferred parameters used in a FASTDB amino acid alignmentare: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20,Randomization Group Length=0, Cutoff Score=1, Window Size=sequencelength, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or thelength of the subject amino acid sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, the results, inpercent identity, must be manually corrected. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query amino acid residues outside the farthest N-and C-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not match/align with the first 10 residues at theN-terminus. The 10 unpaired residues represent 10% of the sequence(number of residues at the N- and C-termini not matched/total number ofresidues in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90residues were perfectly matched the final percent identity would be 90%.In another example, a 90 residue subject sequence is compared with a 100residue query sequence. This time the deletions are internal so thereare no residues at the N- or C-termini of the subject sequence which arenot matched/aligned with the query. In this case the percent identitycalculated by FASTDB is not manually corrected. Once again, only residuepositions outside the N- and C-terminal ends of the subject sequence, asdisplayed in the FASTDB alignment, which are not matched/aligned withthe query sequence are manually corrected. No other manual correctionsare to made for the purposes of the present invention.

The above polypeptide sequences are included irrespective of whetherthey have their normal biological activity. This is because even where aparticular polypeptide molecule does not have biological activity, oneof skill in the art would still know how to use the polypeptide, forinstance, as a vaccine or to generate antibodies. Other uses of thepolypeptides of the present invention that do not have S. aureusactivity include, inter alia, as epitope tags, in epitope mapping, andas molecular weight markers on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods known to those of skill in the art.

As described below, the polypeptides of the present invention can alsobe used to raise polyclonal and monoclonal antibodies, which are usefulin assays for detecting S. aureus protein expression or as agonists andantagonists capable of enhancing or inhibiting S. aureus proteinfunction. Further, such polypeptides can be used in the yeast two-hybridsystem to “capture” S. aureus protein binding proteins which are alsocandidate agonists and antagonists according to the present invention.See, e.g., Fields et al. (1989) Nature 340:245-246.

Epitope-Bearing Portions

In another aspect, the invention provides peptides and polypeptidescomprising epitope-bearing portions of the polypeptides of the presentinvention. These epitopes are immunogenic or antigenic epitopes of thepolypeptides of the present invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response in vivowhen the whole polypeptide of the present invention, or fragmentthereof, is the immunogen. On the other hand, a region of a polypeptideto which an antibody can bind is defined as an “antigenic determinant”or “antigenic epitope.” The number of in vivo immunogenic epitopes of aprotein generally is less than the number of antigenic epitopes. See,e.g., Geysen, et al. (1983) Proc. Natl. Acad. Sci. USA 81:3998-4002.However, antibodies can be made to any antigenic epitope, regardless ofwhether it is an immunogenic epitope, by using methods such as phagedisplay. See e.g., Petersen G. et al. (1995) Mol. Gen. Genet.249:425-431. Therefore, included in the present invention are bothimmunogenic epitopes and antigenic epitopes.

A list of exemplified amino acid sequences comprising immunogenicepitopes of the invention are described herein. It is pointed out thatthese descriptions only lists amino acid residues comprising epitopespredicted to have the highest degree of antigenicity using the algorithmof Jameson and Wolf, (1988) Comp. Appl. Biosci. 4:181-186 (saidreferences incorporated by reference in their entireties). TheJameson-Wolf antigenic analysis was performed using the computer programPROTEAN, using default parameters (Version 3.11 for the Power MacIntosh,DNASTAR, Inc., 1228 South Park Street Madison, Wis.). Amino acidresidues comprising other immunogenic epitopes may be routinelydetermined using algorithms similar to the Jameson-Wolf analysis or byin vivo testing for an antigenic response using methods known in theart. See, e.g., Geysen et al., supra; U.S. Pat. Nos. 4,708,781;5,194,392; 4,433,092; and 5,480,971 (said references incorporated byreference in their entireties).

It is particularly pointed out that the described epitopic amino acidsequences comprise immunogenic epitopes. Table 2 lists only the criticalresidues of immunogenic epitopes determined by the Jameson-Wolfanalysis. Thus, additional flanking residues on either the N-terminal,C-terminal, or both N- and C-terminal ends may be added to the sequencesto generate an epitope-bearing polypeptide of the present invention.Therefore, the immunogenic epitopes may include additional N-terminal orC-terminal amino acid residues. The additional flanking amino acidresidues may be contiguous flanking N-terminal and/or C-terminalsequences from the polypeptides of the present invention, heterologouspolypeptide sequences, or may include both contiguous flanking sequencesfrom the polypeptides of the present invention and heterologouspolypeptide sequences.

Polypeptides of the present invention comprising immunogenic orantigenic epitopes are at least 7 amino acids residues in length. “Atleast” means that a polypeptide of the present invention comprising animmunogenic or antigenic epitope may be 7 amino acid residues in lengthor any integer between 7 amino acids and the number of amino acidresidues of the full length polypeptides of the invention. Preferredpolypeptides comprising immunogenic or antigenic epitopes are at least10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,or 100 amino acid residues in length. However, it is pointed out thateach and every integer between 7 and the number of amino acid residuesof the full length polypeptide are included in the present invention.

The immuno and antigenic epitope-bearing fragments may be specified byeither the number of contiguous amino acid residues, as described above,or further specified by N-terminal and C-terminal positions of thesefragments on the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12,14, 16, 18, 20, or 22. Every combination of a N-terminal and C-terminalposition that a fragment of, for example, at least 7 or at least 15contiguous amino acid residues in length could occupy on the amino acidsequence of SEQ ID NO:2 is included in the invention. Again, “at least 7contiguous amino acid residues in length” means 7 amino acid residues inlength or any integer between 7 amino acids and the number of amino acidresidues of the full length polypeptide of the present invention.Specifically, each and every integer between 7 and the number of aminoacid residues of the full length polypeptide are included in the presentinvention.

Immunogenic and antigenic epitope-bearing polypeptides of the inventionare useful, for example, to make antibodies which specifically bind thepolypeptides of the invention, and in immunoassays to detect thepolypeptides of the present invention. The antibodies are useful, forexample, in affinity purification of the polypeptides of the presentinvention. The antibodies may also routinely be used in a variety ofqualitative or quantitative immuno assays, specifically for thepolypeptides of the present invention using methods known in the art.See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press; 2nd Ed. 1988).

The epitope-bearing polypeptides of the present invention may beproduced by any conventional means for making polypeptides includingsynthetic and recombinant methods known in the art. For instance,epitope-bearing peptides may be synthesized using known methods ofchemical synthesis. For instance, Houghten has described a simple methodfor the synthesis of large numbers of peptides, such as 10-20 mgs of 248individual and distinct 13 residue peptides representing single aminoacid variants of a segment of the HA1 polypeptide, all of which wereprepared and characterized (by ELISA-type binding studies) in less thanfour weeks (Houghten, R. A. Proc. Natl. Acad. Sci. USA 82:5131-5135(1985)). This “Simultaneous Multiple Peptide Synthesis (SMPS)” processis further described in U.S. Pat. No. 4,631,211 to Houghten andcoworkers (1986). In this procedure the individual resins for thesolid-phase synthesis of various peptides are contained in separatesolvent-permeable packets, enabling the optimal use of the manyidentical repetitive steps involved in solid-phase methods. A completelymanual procedure allows 500-1000 or more syntheses to be conductedsimultaneously (Houghten et al. (1985) Proc. Natl. Acad. Sci.82:5131-5135 at 5134.

Epitope-bearing polypeptides of the present invention are used to induceantibodies according to methods well known in the art including, but notlimited to, in vivo immunization, in vitro immunization, and phagedisplay methods. See, e.g., Sutcliffe, et al., supra; Wilson, et al.,supra, and Bittle, et al. (1985) J. Gen. Virol. 66:2347-2354. If in vivoimmunization is used, animals may be immunized with free peptide;however, anti-peptide antibody titer may be boosted by coupling of thepeptide to a macromolecular carrier, such as keyhole limpet hemacyanin(KLH) or tetanus toxoid. For instance, peptides containing cysteineresidues may be coupled to a carrier using a linker such as-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as rabbits, rats and mice are immunizedwith either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μgs of peptide or carrier protein and Freund's adjuvant.Several booster injections may be needed, for instance, at intervals ofabout two weeks, to provide a useful titer of anti-peptide antibodywhich can be detected, for example, by ELISA assay using free peptideadsorbed to a solid surface. The titer of anti-peptide antibodies inserum from an immunized animal may be increased by selection ofanti-peptide antibodies, for instance, by adsorption to the peptide on asolid support and elution of the selected antibodies according tomethods well known in the art.

As one of skill in the art will appreciate, and discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to heterologous polypeptide sequences.For example, the polypeptides of the present invention may be fused withthe constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, any combination thereof including both entiredomains and portions thereof) resulting in chimeric polypeptides. Thesefusion proteins facilitate purification, and show an increased half-lifein vivo. This has been shown, e.g., for chimeric proteins consisting ofthe first two domains of the human CD4-polypeptide and various domainsof the constant regions of the heavy or light chains of mammalianimmunoglobulins. See, e.g., EPA 0,394,827; Traunecker et al. (1988)Nature 331:84-86. Fusion proteins that have a disulfide-linked dimericstructure due to the IgG portion can also be more efficient in bindingand neutralizing other molecules than monomeric polypeptides orfragments thereof alone. See, e.g., Fountoulakis et al. (1995) J.Biochem. 270:3958-3964. Nucleic acids encoding the above epitopes canalso be recombined with a gene of interest as an epitope tag to aid indetection and purification of the expressed polypeptide.

TABLE 2 Residues Comprising Antigenic Epitope-Bearing Portion. Fem XAbout Asn-30 to about Lys-34; about Lys-45 to about Glu-50; about Asn-99to about Gly- 101; about Tyr-148 to about Gln-153; about Asn-181 toabout Lys-184; about Asn-272 to about Ser-275; about Asn-278 to aboutLys-281; about Arg-295 to about Lys-298; about Ser-331 to about Tyr-335;and about Lys-398 to about Val-400, all of SEQ ID NO:2. fur A AboutAsp-45 to about Tyr-48; and about Lys-96 to about Asp-99, all of SEQ IDNO:4. fur B About Pro-28 to about Arg-30; about Pro-44 to about Ala-46;about Asp-85 to about Arg- 89; about Phe-90 to about Phe-92; aboutGln-104 to about Gly-106; and about Cys-145 to about Lys-148, all of SEQID NO:6. fur C About Thr-20 to about Arg-23; about Phe-77 to aboutGly-80; and about Ala-145 to about Ter-150, all of SEQ ID NO:8. fmt BAbout Ser-29 to about Gln-40; about Leu-51 to about His-55; about Gln-78to about Lys- 82; about Tyr-132 to about Gln-135; about Ile-155 to aboutSer-157; about Val-161 to about Arg-167; about Lys-183 to about Glu-186;about Thr-232 to about Asp-239; about Lys-254 to about Glu-256; aboutPro-284 to about Lys-289; about Ser-295 to about Leu- 299; about Glu-305to about Ala-308; about Asn-318 to about Glu-321; about Pro-341 to aboutAsn-344; about Lys-373 to about Ser-376; about Gln-384 to about Asp-387;and about Arg-422 to about Arg-426, all of SEQ ID NO:10. pbpF AboutGlu-7 to about Asp-11; about Ile-18 to about Lys-20; about Ile-55 toabout Glu-59; about Ser-66 to about Gly-77; about Thr-92 to aboutThr-97; about Arg-123 to about Asp- 127; about Lys-154 to about Asp-159;about Ile-166 to about Gln-170; about Arg-230 to about Tyr-234; aboutThr-247 to about Gly-251; about Lys-263 to about Ser-274; about Thr-294to about Lys-300; about Gly-310 to about Leu-316; about Ser-341 to aboutAsp- 346; about Ile-370 to about Asp-378; about Ser-393 to aboutGly-396; about Leu-425 to about Lys-427; about Lys-433 to about Gly-435;about Pro-478 to about Gly-485; about Leu-499 to about Gly-505; aboutThr-511 to about Pro-514; about Asp-543 to about Tyr 545; about Ser-558to about Glu-563; about Asn-598 to about Gly-602; Lys-618 to aboutThr-621; about Gly-628 to about Val-632; about Asp-643 to about Lys-646;about Asp- 667 to about Arg-670; about Gly-681 to about Asp-684; andabout Asn-686 to about Lys- 689, all of SEQ ID NO:12. pbpG About Gln-4to about His-8; about Lys-55 to about Ser-59; about Ile-72 to aboutAsp-75; about Lys-101 to about Ser-104; about Asp-142 to about Ser-145;about Asp-201 to about Gly-204; about Pro-221 to about Asp-224; aboutPro-245 to about Asp-249; about Lys- 253 to about Lys-256; and aboutAsn-297 to about Ter-302, all of SEQ ID NO:14. cbrA About Asn-23 toabout Lys-35; about Ile-49 to about Lys-54; about Pro-85 to about Glu-88; about Asp-233 to about Lys-236; about Ser-243 to about Ile-247;about Ser-260 to about Ala-264; about Asp-296 to about Leu-298; andabout Tyr-309 to about Ser-312, all of SEQ ID NO:16. cbr B About Asp-44to about Asn-47; about Thr-219 to about Asp-222; and about Lys-325 toabout Arg-328, all of SEQ ID NO:18. cbrC About Asn-48 to about Asp-52;and about Lys-141 to about Arg-145, all of SEQ ID NO:20. enolase AboutLeu 13 to about Pro 19; about Gly 63 to about Thr 65; about Thr 102 toabout Lys 107; about Ser 156 to about Asp 159 about Arg 198 to about Leu200; about Asp 206 to about Gly 209; about Lys 234 to about Glu 237;about Tyr 25 to about Asp 257; about Met 294 to about Gly 301; about Arg308 to about Asp 311; about His 371 to about Glu 375; about Ser 397 toabout Asp 402; about Lys 422 to about Gly 425, all of SEQ ID NO:22.

Antibodies

The present invention further relates to antibodies and T-cell antigenreceptors (TCR) which specifically bind the polypeptides of the presentinvention. The antibodies of the present invention include IgG(including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2),IgD, IgE, or IgM, and IgY. As used herein, the term “antibody” (Ab) ismeant to include whole antibodies, including single-chain wholeantibodies, and antigen-binding fragments thereof. Most preferably theantibodies are human antigen binding antibody fragments of the presentinvention include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv) and fragments comprising either a V_(L) or V_(H) domain. Theantibodies may be from any animal origin including birds and mammals.Preferably, the antibodies are human, murine, rabbit, goat, guinea pig,camel, horse, or chicken.

Antigen-binding antibody fragments, including single-chain antibodies,may comprise the variable region(s) alone or in combination with theentire or partial of the following: hinge region, CH1, CH2, and CH3domains. Also included in the invention are any combinations of variableregion(s) and hinge region, CH1, CH2, and CH3 domains. The presentinvention further includes chimeric, humanized, and human monoclonal andpolyclonal antibodies which specifically bind the polypeptides of thepresent invention. The present invention further includes antibodieswhich are anti-idiotypic to the antibodies of the present invention.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for heterologous compositions, such as aheterologous polypeptide or solid support material. See, e.g., WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al. (1991)J. Immunol. 147:60-69; U.S. Pat. Nos. 5,573,920, 4,474,893, 5,601,819,4,714,681, 4,925,648; Kostelny, S. A. et al. (1992) J. Immunol.148:1547-1553.

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which are recognized or specifically bound by the antibody.The epitope(s) or polypeptide portion(s) may be specified as describedherein, e.g., by N-terminal and C-terminal positions, by size incontiguous amino acid residues, or listed in the Tables and Figures.Antibodies which specifically bind any epitope or polypeptide of thepresent invention may also be excluded. Therefore, the present inventionincludes antibodies that specifically bind polypeptides of the presentinvention, and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of the polypeptides of the presentinvention are included. Antibodies that do not bind polypeptides withless than 95%, less than 90%, less than 85%, less than 80%, less than75%, less than 70%, less than 65%, less than 60%, less than 55%, andless than 50% identity (as calculated using methods known in the art anddescribed herein) to a polypeptide of the present invention are alsoincluded in the present invention. Further included in the presentinvention are antibodies which only bind polypeptides encoded bypolynucleotides which hybridize to a polynucleotide of the presentinvention under stringent hybridization conditions (as describedherein). Antibodies of the present invention may also be described orspecified in terms of their binding affinity. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M,5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M,5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M, and 10⁻¹⁵M.

Antibodies of the present invention have uses that include, but are notlimited to, methods known in the art to purify, detect, and target thepolypeptides of the present invention including both in vitro and invivo diagnostic and therapeutic methods. For example, the antibodieshave use in immunoassays for qualitatively and quantitatively measuringlevels of the polypeptides of the present invention in biologicalsamples. See, e.g., Harlow et al., ANTIBODIES: A LABORATORY MANUAL,(Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated byreference in the entirety).

The antibodies of the present invention may be used either alone or incombination with other compositions. The antibodies may further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalently andnon-covalently conjugations) to polypeptides or other compositions. Forexample, antibodies of the present invention may be recombinantly fusedor conjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs, or toxins.See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 0 396 387.

The antibodies of the present invention may be prepared by any suitablemethod known in the art. For example, a polypeptide of the presentinvention or an antigenic fragment thereof can be administered to ananimal in order to induce the production of sera containing polyclonalantibodies. Monoclonal antibodies can be prepared using a wide oftechniques known in the art including the use of hybridoma andrecombinant technology. See, e.g., Harlow et al., ANTIBODIES: ALABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling, et al., in: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS563-681 (Elsevier, N.Y., 1981) (said references incorporated byreference in their entireties).

Fab and F(ab′)2 fragments may be produced by proteolytic cleavage, usingenzymes such as papain (to produce Fab fragments) or pepsin (to produceF(ab′)2 fragments).

Alternatively, antibodies of the present invention can be producedthrough the application of recombinant DNA technology or throughsynthetic chemistry using methods known in the art. For example, theantibodies of the present invention can be prepared using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of a phage particle whichcarries polynucleotide sequences encoding them. Phage with a desiredbinding property are selected from a repertoire or combinatorialantibody library (e.g. human or murine) by selecting directly withantigen, typically antigen bound or captured to a solid surface or bead.Phage used in these methods are typically filamentous phage including fdand M13 with Fab, Fv or disulfide stabilized Fv antibody domainsrecombinantly fused to either the phage gene III or gene VIII protein.Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in BrinkmanU. et al. (1995) J. Immunol. Methods 182:41-50; Ames, R. S. et al.(1995) J. Immunol. Methods 184:177-186; Kettleborough, C. A. et al.(1994) Eur. J. Immunol. 24:952-958; Persic, L. et al. (1997) Gene 1879-18; Burton, D. R. et al. (1994) Advances in Immunology 57:191-280;PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426,5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743(said references incorporated by reference in their entireties).

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired hostincluding mammalian cells, insect cells, plant cells, yeast, andbacteria. For example, techniques to recombinantly produce Fab, Fab′ andF(ab′)2 fragments can also be employed using methods known in the artsuch as those disclosed in WO 92/22324; Mullinax, R. L. et al. (1992)BioTechniques 12(6):864-869; and Sawai, H. et al. (1995) AJRI 34:26-34;and Better, M. et al. (1988) Science 240:1041-1043 (said referencesincorporated by reference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al. (1991) Methods in Enzymology 203:46-88; Shu, L.et al. (1993) PNAS 90:7995-7999; and Skerra, A. et al. (1988) Science240:1038-1040. For some uses, including in vivo use of antibodies inhumans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. Methods for producing chimericantibodies are known in the art. See e.g., Morrison, Science 229:1202(1985); Oi et al., BioTechniques 4:214 (1986); Gillies, S. D. et al.(1989) J. Immunol. Methods 125:191-202; and U.S. Pat. No. 5,807,715.Antibodies can be humanized using a variety of techniques includingCDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101; and5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596; PadlanE. A., (1991) Molecular Immunology 28(4/5):489-498; Studnicka G. M. etal. (1994) Protein Engineering 7(6):805-814; Roguska M. A. et al. (1994)PNAS 91:969-973), and chain shuffling (U.S. Pat. No. 5,565,332). Humanantibodies can be made by a variety of methods known in the artincluding phage display methods described above. See also, U.S. Pat.Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and WO 98/46645(said references incorporated by reference in their entireties).

Further included in the present invention are antibodies recombinantlyfused or chemically conjugated (including both covalently andnon-covalently conjugations) to a polypeptide of the present invention.The antibodies may be specific for antigens other than polypeptides ofthe present invention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal. supra and WO 93/21232; EP 0 439 095; Naramura, M. et al. (1994)Immunol. Lett. 39:91-99; U.S. Pat. No. 5,474,981; Gillies, S. O. et al.(1992) PNAS 89:1428-1432; Fell, H. P. et al. (1991) J. Immunol.146:2446-2452 (said references incorporated by reference in theirentireties).

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Feregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the hinge region, CH1 domain, CH2domain, and CH3 domain or any combination of whole domains or portionsthereof. The polypeptides of the present invention may be fused orconjugated to the above antibody portions to increase the in vivo halflife of the polypeptides or for use in immunoassays using methods knownin the art. The polypeptides may also be fused or conjugated to theabove antibody portions to form multimers. For example, Fe portionsfused to the polypeptides of the present invention can form dimersthrough disulfide bonding between the Fc portions. Higher multimericforms can be made by fusing the polypeptides to portions of IgA and IgM.Methods for fusing or conjugating the polypeptides of the presentinvention to antibody portions are known in the art. See e.g., U.S. Pat.Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946;EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi, A. etal. (1991) PNAS 88:10535-10539; Zheng, X. X. et al. (1995) J. Immunol.154:5590-5600; and Vil, H. et al. (1992) PNAS 89:11337-11341 (saidreferences incorporated by reference in their entireties).

The invention further relates to antibodies which act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies which disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. Included are both receptor-specificantibodies and ligand-specific antibodies. Included arereceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. Also include are receptor-specific antibodies which both preventligand binding and receptor activation. Likewise, included areneutralizing antibodies which bind the ligand and prevent binding of theligand to the receptor, as well as antibodies which bind the ligand,thereby preventing receptor activation, but do not prevent the ligandfrom binding the receptor. Further included are antibodies whichactivate the receptor. These antibodies may act as agonists for eitherall or less than all of the biological activities affected byligand-mediated receptor activation. The antibodies may be specified asagonists or antagonists for biological activities comprising specificactivities disclosed herein. The above antibody agonists can be madeusing methods known in the art. See e.g., WO 96/40281; U.S. Pat. No.5,811,097; Deng, B. et al. (1998) Blood 92(6):1981-1988; Chen, Z. et al.(1998) Cancer Res. 58(16):3668-3678; Harrop, J. A. et al. (1998) J.Immunol. 161(4):1786-1794; Zhu, Z. et al. (1998) Cancer Res.58(15):3209-3214; Yoon, D. Y. et al. (1998) J. Immunol.160(7):3170-3179; Prat, M. et al. (1998) J. Cell. Sci. 111(Pt2):237-247;Pitard, V. et al (1997) J. Immunol. Methods 205(2):177-190; Liautard, J.et al. (1997) Cytokinde 9(4):233-241; Carlson, N. G. et al. (1997) J.Biol. Chem. 272(17):11295-11301; Taryman, R. E. et al. (1995) Neuron14(4):755-762; Muller, Y. A. et al. (1998) Structure 6(9):1153-1167;Bartunek, P. et al. (1996) Cytokine 8(1):14-20 (said referencesincorporated by reference in their entireties).

Diagnostic Assays

The present invention further relates to methods for assayingstaphylococcal infection in an animal by detecting the expression ofgenes encoding staphylococcal polypeptides of the present invention. Themethods comprise analyzing tissue or body fluid from the animal forStaphylococcus-specific antibodies, nucleic acids, or proteins. Analysisof nucleic acid specific to Staphylococcus is assayed by PCR orhybridization techniques using nucleic acid sequences of the presentinvention as either hybridization probes or primers. See, e.g., Sambrooket al. Molecular cloning: A Laboratory Manual (Cold Spring HarborLaboratory Press, 2nd ed., 1989, page 54 reference); Eremeeva et al.(1994) J. Clin. Microbiol. 32:803-810 (describing differentiation amongspotted fever group Rickettsiae species by analysis of restrictionfragment length polymorphism of PCR-amplified DNA) and Chen et al. 1994J. Clin. Microbiol. 32:589-595 (detecting B. burgdorferi nucleic acidsvia PCR).

Where diagnosis of a disease state related to infection withStaphylococcus has already been made, the present invention is usefulfor monitoring progression or regression of the disease state wherebypatients exhibiting enhanced Staphylococcus gene expression willexperience a worse clinical outcome relative to patients expressingthese gene(s) at a lower level.

By “biological sample” is intended any biological sample obtained froman animal, cell line, tissue culture, or other source which containsStaphylococcus polypeptide, mRNA, or DNA. Biological samples includebody fluids (such as saliva, blood, plasma, urine, mucus, synovialfluid, etc.) tissues (such as muscle, skin, and cartilage) and any otherbiological source suspected of containing Staphylococcus polypeptides ornucleic acids. Methods for obtaining biological samples such as tissueare well known in the art.

The present invention is useful for detecting diseases related toStaphylococcus infections in animals. Preferred animals include monkeys,apes, cats, dogs, birds, cows, pigs, mice, horses, rabbits and humans.Particularly preferred are humans.

Total RNA can be isolated from a biological sample using any suitabletechnique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski et al. (1987) Anal. Biochem. 162:156-159. mRNA encodingStaphylococcus polypeptides having sufficient homology to the nucleicacid sequences identified in Table 1 to allow for hybridization betweencomplementary sequences are then assayed using any appropriate method.These include Northern blot analysis, S1 nuclease mapping, thepolymerase chain reaction (PCR), reverse transcription in combinationwith the polymerase chain reaction (RT-PCR), and reverse transcriptionin combination with the ligase chain reaction (RT-LCR).

Northern blot analysis can be performed as described in Harada et al.(1990) Cell 63:303-312. Briefly, total RNA is prepared from a biologicalsample as described above. For the Northern blot, the RNA is denaturedin an appropriate buffer (such as glyoxal/dimethyl sulfoxide/sodiumphosphate buffer), subjected to agarose gel electrophoresis, andtransferred onto a nitrocellulose filter. After the RNAs have beenlinked to the filter by a UV linker, the filter is prehybridized in asolution containing formamide, SSC, Denhardt's solution, denaturedsalmon sperm, SDS, and sodium phosphate buffer. A S. aureus polypeptideDNA sequence shown in Table 1 labeled according to any appropriatemethod (such as the ³²P-multiprimed DNA labeling system (Amersham)) isused as probe. After hybridization overnight, the filter is washed andexposed to x-ray film. DNA for use as probe according to the presentinvention is described in the sections above and will preferably atleast 15 nucleotides in length.

S1 mapping can be performed as described in Fujita et al. (1987) Cell49:357-367. To prepare probe DNA for use in S1 mapping, the sense strandof an above-described S. aureus DNA sequence of the present invention isused as a template to synthesize labeled antisense DNA. The antisenseDNA can then be digested using an appropriate restriction endonucleaseto generate further DNA probes of a desired length. Such antisenseprobes are useful for visualizing protected bands corresponding to thetarget mRNA (i.e., mRNA encoding Staphylococcus polypeptides).

Levels of mRNA encoding Staphylococcus polypeptides are assayed, fore.g., using the RT-PCR method described in Makino et al. (1990)Technique 2:295-301. By this method, the radioactivities of the“amplicons” in the polyacrylamide gel bands are linearly related to theinitial concentration of the target mRNA. Briefly, this method involvesadding total RNA isolated from a biological sample in a reaction mixturecontaining a RT primer and appropriate buffer. After incubating forprimer annealing, the mixture can be supplemented with a RT buffer,dNTPs, DTT, RNase inhibitor and reverse transcriptase. After incubationto achieve reverse transcription of the RNA, the RT products are thensubject to PCR using labeled primers. Alternatively, rather thanlabeling the primers, a labeled dNTP can be included in the PCR reactionmixture. PCR amplification can be performed in a DNA thermal cycleraccording to conventional techniques. After a suitable number of roundsto achieve amplification, the PCR reaction mixture is electrophoresed ona polyacrylamide gel. After drying the gel, the radioactivity of theappropriate bands (corresponding to the mRNA encoding the Staphylococcuspolypeptides of the present invention) are quantified using an imaginganalyzer. RT and PCR reaction ingredients and conditions, reagent andgel concentrations, and labeling methods are well known in the art.Variations on the RT-PCR method will be apparent to the skilled artisan.Other PCR methods that can detect the nucleic acid of the presentinvention can be found in PCR PRIMER: A LABORATORY MANUAL (C. W.Dieffenbach et al. eds., Cold Spring Harbor Lab Press, 1995).

The polynucleotides of the present invention, including both DNA andRNA, may be used to detect polynucleotides of the present invention orStaphylococcal species including S. aureus using bio-chip technology.The present invention includes both high density chip arrays (>1000oligonucleotides per cm²) and low density chip arrays (<1000oligonucleotides per cm²). Bio-chips comprising arrays ofpolynucleotides of the present invention may be used to detectStaphylococcal species, including S. aureus, in biological andenvironmental samples and to diagnose an animal, including humans, withan S. aureus or other Staphylococcal infection. The bio-chips of thepresent invention may comprise polynucleotide sequences of otherpathogens including bacteria, viral, parasitic, and fungalpolynucleotide sequences, in addition to the polynucleotide sequences ofthe present invention, for use in rapid differential pathogenicdetection and diagnosis. The bio-chips can also be used to monitor an S.aureus or other Staphylococcal infections and to monitor the geneticchanges (deletions, insertions, mismatches, etc.) in response to drugtherapy in the clinic and drug development in the laboratory. Thebio-chip technology comprising arrays of polynucleotides of the presentinvention may also be used to simultaneously monitor the expression of amultiplicity of genes, including those of the present invention. Inaddition, the bio-chips of the present invention may be used to screenlarge numbers of peptides, polypeptides, antibodies, small molecules andother drug compounds which bind to the polynucleotides of the presentinvention. The bio-chips may also be used to measure relative binding orbinding affinities (in on-rates or off-rates) of peptides, polypeptides,antibodies, small molecules and other drug compounds to thepolynucleotides of the present invention. The polynucleotides used tocomprise a selected array may be specified in the same manner as for thefragments, i.e., by their 5′ and 3′ positions or length in contiguousbase pairs and include from. Methods and particular uses of thepolynucleotides of the present invention to detect Staphylococcalspecies, including S. aureus, using bio-chip technology include thoseknown in the art and those of: U.S. Pat. Nos. 5,324,633, 5,510,270,5,545,531, 5,445,934, 5,677,195, 5,532,128, 5,556,752, 5,527,681,5,451,683, 5,424,186, 5,607,646, 5,658,732 and World Patent Nos.WO/9710365, WO/9511995, WO/9743447, WO/9535505, each incorporated hereinin their entireties.

Biosensors using the polynucleotides of the present invention may alsobe used to detect, diagnose, and monitor S. aureus or otherStaphylococcal species and infections thereof. Biosensors using thepolynucleotides of the present invention may also be used to detectparticular polynucleotides of the present invention. Biosensors usingthe polynucleotides of the present invention may also be used to monitorthe genetic changes (deletions, insertions, mismatches, etc.) inresponse to drug therapy in the clinic and drug development in thelaboratory. In addition, the biosensors of the present invention may beused to screen large numbers of polynucleotides, peptides, polypeptides,antibodies, small molecules and other drug compounds which bind to thepolynucleotides of the present invention. The biosensors may also beused to measure relative binding or binding affinities (in on-rates oroff-rates) of polynucleotides, peptides, polypeptides, antibodies, smallmolecules, and other drug compounds to the polynucleotides of thepresent invention. Methods and particular uses of the polynucleotides ofthe present invention to detect Staphylococcal species, including S.aureus, using biosensors include those known in the art and those of:U.S. Pat. Nos. 5,721,102, 5,658,732, 5,631,170, and World Patent Nos.WO/9735011, WO/9720203, each incorporated herein in their entireties.

Thus, the present invention includes both bio-chips and biosensorscomprising polynucleotides of the present invention and methods of theiruse.

Assaying Staphylococcus polypeptide levels in a biological sample canoccur using any art-known method, such as antibody-based techniques. Forexample, Staphylococcus polypeptide expression in tissues can be studiedwith classical immunohistological methods. In these, the specificrecognition is provided by the primary antibody (polyclonal ormonoclonal) but the secondary detection system can utilize fluorescent,enzyme, or other conjugated secondary antibodies. As a result, animmunohistological staining of tissue section for pathologicalexamination is obtained. Tissues can also be extracted, e.g., with ureaand neutral detergent, for the liberation of Staphylococcus polypeptidesfor Western-blot or dot/slot assay. See, e.g., Jalkanen, M. et al.(1985) J. Cell. Biol. 101:976-985; Jalkanen, M. et al. (1987) J. Cell .Biol. 105:3087-3096. In this technique, which is based on the use ofcationic solid phases, quantitation of a Staphylococcus polypeptide canbe accomplished using an isolated Staphylococcus polypeptide as astandard. This technique can also be applied to body fluids.

Other antibody-based methods useful for detecting Staphylococcuspolypeptide gene expression include immunoassays, such as the ELISA andthe radioimmunoassay (RIA). For example, a Staphylococcuspolypeptide-specific monoclonal antibodies can be used both as animmunoabsorbent and as an enzyme-labeled probe to detect and quantify aStaphylococcus polypeptide. The amount of a Staphylococcus polypeptidepresent in the sample can be calculated by reference to the amountpresent in a standard preparation using a linear regression computeralgorithm. Such an ELISA is described in lacobelli et al. (1988) BreastCancer Research and Treatment 11:19-30. In another ELISA assay, twodistinct specific monoclonal antibodies can be used to detectStaphylococcus polypeptides in a body fluid. In this assay, one of theantibodies is used as the immunoabsorbent and the other as theenzyme-labeled probe.

The above techniques may be conducted essentially as a “one-step” or“two-step” assay. The “one-step” assay involves contacting theStaphylococcus polypeptide with immobilized antibody and, withoutwashing, contacting the mixture with the labeled antibody. The“two-step” assay involves washing before contacting the mixture with thelabeled antibody. Other conventional methods may also be employed assuitable. It is usually desirable to immobilize one component of theassay system on a support, thereby allowing other components of thesystem to be brought into contact with the component and readily removedfrom the sample. Variations of the above and other immunological methodsincluded in the present invention can also be found in Harlow et al.,ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press,2nd ed. 1988).

Suitable enzyme labels include, for example, those from the oxidasegroup, which catalyze the production of hydrogen peroxide by reactingwith substrate. Glucose oxidase is particularly preferred as it has goodstability and its substrate (glucose) is readily available. Activity ofan oxidase label may be assayed by measuring the concentration ofhydrogen peroxide formed by the enzyme-labeled antibody/substratereaction. Besides enzymes, other suitable labels include radioisotopes,such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulphur (³⁵S), tritium (³H),indium (¹¹²In), and technetium (^(99m)Tc), and fluorescent labels, suchas fluorescein and rhodamine, and biotin.

Further suitable labels for the Staphylococcus polypeptide-specificantibodies of the present invention are provided below. Examples ofsuitable enzyme labels include malate dehydrogenase, staphylococcalnuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase,alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase,peroxidase, alkaline phosphatase, asparaginase, glucose oxidase,beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphatedehydrogenase, glucoamylase, and acetylcholine esterase.

Examples of suitable radioisotopic labels include ³H, ¹¹¹In, ¹²⁵I, ¹³¹I,³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci,²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, etc. ¹¹¹In is a preferred isotope where invivo imaging is used since its avoids the problem of dehalogenation ofthe ¹²⁵I or ¹³¹I-labeled monoclonal antibody by the liver. In addition,this radionucleotide has a more favorable gamma emission energy forimaging. See, e.g., Perkins et al. (1985) Eur. J. Nucl. Med. 10:296-301;Carasquillo et al. (1987) J. Nucl. Med. 28:281-287. For example, ¹¹¹Incoupled to monoclonal antibodies with 1-(P-isothiocyanatobenzyl)-DPTAhas shown little uptake in non-tumors tissues, particularly the liver,and therefore enhances specificity of tumor localization. See, Estebanet al. (1987) J. Nucl. Med. 28:861-870.

Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd,⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, and ⁵⁶Fe.

Examples of suitable fluorescent labels include an ¹⁵²Eu label, afluorescein label, an isothiocyanate label, a rhodamine label, aphycoerythrin label, a phycocyanin label, an allophycocyanin label, ano-phthaldehyde label, and a fluorescamine label.

Examples of suitable toxin labels include, Pseudomonas toxin, diphtheriatoxin, ricin, and cholera toxin.

Examples of chemiluminescent labels include a luminal label, anisoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridinium salt label, an oxalate ester label, a luciferinlabel, a luciferase label, and an aequorin label.

Examples of nuclear magnetic resonance contrasting agents include heavymetal nuclei such as Gd, Mn, and iron.

Typical techniques for binding the above-described labels to antibodiesare provided by Kennedy et al. (1976) Clin. Chim. Acta 70:1-31, andSchurs et al. (1977) Clin. Chim. Acta 81:1-40. Coupling techniquesmentioned in the latter are the glutaraldehyde method, the periodatemethod, the dimaleimide method, them-maleimidobenzyl-N-hydroxy-succinimide ester method, all of whichmethods are incorporated by reference herein.

In a related aspect, the invention includes a diagnostic kit for use inscreening serum containing antibodies specific against S. aureusinfection. Such a kit may include an isolated S. aureus antigencomprising an epitope which is specifically immunoreactive with at leastone anti-S. aureus antibody. Such a kit also includes means fordetecting the binding of said antibody to the antigen. In specificembodiments, the kit may include a recombinantly produced or chemicallysynthesized peptide or polypeptide antigen. The peptide or polypeptideantigen may be attached to a solid support.

In a more specific embodiment, the detecting means of theabove-described kit includes a solid support to which said peptide orpolypeptide antigen is attached. Such a kit may also include anon-attached reporter-labeled anti-human antibody. In this embodiment,binding of the antibody to the S. aureus antigen can be detected bybinding of the reporter labeled antibody to the anti-S. aureuspolypeptide antibody.

In a related aspect, the invention includes a method of detecting S.aureus infection in a subject. This detection method includes reacting abody fluid, preferably serum, from the subject with an isolated S.aureus antigen, and examining the antigen for the presence of boundantibody. In a specific embodiment, the method includes a polypeptideantigen attached to a solid support, and serum is reacted with thesupport. Subsequently, the support is reacted with a reporter-labeledanti-human antibody. The support is then examined for the presence ofreporter-labeled antibody.

The solid surface reagent employed in the above assays and kits isprepared by known techniques for attaching protein material to solidsupport material, such as polymeric beads, dip sticks, 96-well plates orfilter material. These attachment methods generally include non-specificadsorption of the protein to the support or covalent attachment of theprotein , typically through a free amine group, to a chemically reactivegroup on the solid support, such as an activated carboxyl, hydroxyl, oraldehyde group. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

The polypeptides and antibodies of the present invention, includingfragments thereof, may be used to detect Staphylococcal speciesincluding S. aureus using bio-chip and biosensor technology. Bio-chipand biosensors of the present invention may comprise the polypeptides ofthe present invention to detect antibodies, which specifically recognizeStaphylococcal species, including S. aureus. Bio-chip and biosensors ofthe present invention may also comprise antibodies which specificallyrecognize the polypeptides of the present invention to detectStaphylococcal species, including S. aureus or specific polypeptides ofthe present invention. Bio-chips or biosensors comprising polypeptidesor antibodies of the present invention may be used to detectStaphylococcal species, including S. aureus, in biological andenvironmental samples and to diagnose an animal, including humans, withan S. aureus or other Staphylococcal infection. Thus, the presentinvention includes both bio-chips and biosensors comprising polypeptidesor antibodies of the present invention and methods of their use.

The bio-chips of the present invention, discussed above, may furthercomprise polypeptide sequences of other pathogens including bacteria,viral, parasitic, and fungal polypeptide sequences, in addition to thepolypeptide sequences of the present invention, for use in rapiddifferential pathogenic detection and diagnosis. The bio-chips of thepresent invention may further comprise antibodies or fragments thereofspecific for other pathogens including bacteria, viral, parasitic, andfungal polypeptide sequences, in addition to the antibodies or fragmentsthereof of the present invention, for use in rapid differentialpathogenic detection and diagnosis. The bio-chips and biosensors of thepresent invention may also be used to monitor an S. aureus or otherStaphylococcal infection and to monitor the genetic changes (amino aciddeletions, insertions, substitutions, etc.) in response to drug therapyin the clinic and drug development in the laboratory. The bio-chip andbiosensors comprising polypeptides or antibodies of the presentinvention may also be used to simultaneously monitor the expression of amultiplicity of polypeptides, including those of the present invention.In addition, the bio-chips and biosensors of the present invention maybe used to screen large numbers of polynucleotides, peptides,polypeptides, antibodies, small molecules, and other drug compoundswhich bind to the polypeptides of the present invention. The bio-chipsmay also be used to measure relative binding or binding affinities (inon-rates or off-rates) of polynucleotides, peptides, polypeptides,antibodies, small molecules and other drug compounds to the polypeptidesof the present invention. The polypeptides used to comprise a bio-chipor biosensor of the present invention may be specified in the samemanner as for the fragments, i.e., by their N-terminal and C-terminalpositions or length in contiguous amino acid residue. Methods andparticular uses of the polypeptides and antibodies of the presentinvention to detect Staphylococcal species, including S. aureus, orspecific polypeptides using bio-chip and biosensor technology includethose known in the art, those of the U.S. Pat. Nos. and World PatentNos. listed above for bio-chips and biosensors using polynucleotides ofthe present invention, and those of: U.S. Pat. Nos. 5,324,633,5,658,732, 5,135,852, 5,567,301, 5,677,196, 5,690,894, 5,527,681,5,510,270, 5,545,531, 5,445,934, 5,677,195, 5,532,128, 5,556,752,5,451,683, 5,424,186, 5,607,646, and World Patent Nos. WO/9729366,WO/9612957, WO/9710365, WO/9511995, WO/9743447, WO/9535505, eachincorporated herein in their entireties.

Treatment

Agonists and Antagonists—Assays and Molecules

The invention also provides a method of screening compounds to identifythose which enhance or block the biological activity of the S. aureuspolypeptides of the present invention. The present invention furtherprovides where the compounds kill or slow the growth of S. aureus. Theability of S. aureus antagonists, including S. aureus ligands, toprophylactically or therapeutically block antibiotic resistance may beeasily tested by the skilled artisan. See, e.g., Straden et al. (1997) JBacteriol. 179(1):9-16.

An agonist is a compound which increases the natural biological functionor which functions in a manner similar to the polypeptides of thepresent invention, while antagonists decrease or eliminate suchfunctions. Potential antagonists include small organic molecules,peptides, polypeptides, and antibodies that bind to a polypeptide of theinvention and thereby inhibit or extinguish its activity.

The antagonists may be employed for instance to inhibit peptidoglycancross bridge formation. Antibodies against S. aureus may be employed tobind to and inhibit S. aureus activity to treat antibiotic resistance.Any of the above antagonists may be employed in a composition with apharmaceutically acceptable carrier.

Vaccines

The present invention also provides vaccines comprising one or morepolypeptides of the present invention. Heterogeneity in the compositionof a vaccine may be provided by combining S. aureus polypeptides of thepresent invention. Multi-component vaccines of this type are desirablebecause they are likely to be more effective in eliciting protectiveimmune responses against multiple species and strains of theStaphylococcus genus than single polypeptide vaccines.

Multi-component vaccines are known in the art to elicit antibodyproduction to numerous immunogenic components. See, e.g., Decker et al.(1996) J. Infect. Dis. 174:S270-275. In addition, a hepatitis B,diphtheria, tetanus, pertussis tetravalent vaccine has recently beendemonstrated to elicit protective levels of antibodies in human infantsagainst all four pathogenic agents. See, e.g., Aristegui, J. et al.(1997) Vaccine 15:7-9.

The present invention in addition to single-component vaccines includesmulti-component vaccines. These vaccines comprise more than onepolypeptide, immunogen or antigen. Thus, a multi-component vaccine wouldbe a vaccine comprising more than one of the S. aureus polypeptides ofthe present invention.

Further within the scope of the invention are whole cell and whole viralvaccines. Such vaccines may be produced recombinantly and involve theexpression of one or more of the S. aureus polypeptides described inTable 1. For example, the S. aureus polypeptides of the presentinvention may be either secreted or localized intracellular, on the cellsurface, or in the periplasmic space. Further, when a recombinant virusis used, the S. aureus polypeptides of the present invention may, forexample, be localized in the viral envelope, on the surface of thecapsid, or internally within the capsid. Whole cells vaccines whichemploy cells expressing heterologous proteins are known in the art. See,e.g., Robinson, K. et al. (1997) Nature Biotech. 15:653-657; Sirard, J.et al. (1997) Infect. Immun. 65:2029-2033; Chabalgoity, J. et al. (1997)Infect. Immun. 65:2402-2412. These cells may be administered live or maybe killed prior to administration. Chabalgoity, J. et al., supra, forexample, report the successful use in mice of a live attenuatedSalmonella vaccine strain which expresses a portion of a platyhelminthfatty acid-binding protein as a fusion protein on its cells surface.

A multi-component vaccine can also be prepared using techniques known inthe art by combining one or more S. aureus polypeptides of the presentinvention, or fragments thereof, with additional non-staphylococcalcomponents (e.g., diphtheria toxin or tetanus toxin, and/or othercompounds known to elicit an immune response). Such vaccines are usefulfor eliciting protective immune responses to both members of theStaphylococcus genus and non-staphylococcal pathogenic agents.

The vaccines of the present invention also include DNA vaccines. DNAvaccines are currently being developed for a number of infectiousdiseases. See, et al., Boyer, et al. (1997) Nat. Med. 3:526-532;reviewed in Spier, R. (1996) Vaccine 14:1285-1288. Such DNA vaccinescontain a nucleotide sequence encoding one or more S. aureuspolypeptides of the present invention oriented in a manner that allowsfor expression of the subject polypeptide. For example, the directadministration of plasmid DNA encoding B. burgdorgeri OspA has beenshown to elicit protective immunity in mice against borrelial challenge.See, Luke et al. (1997) J. Infect. Dis. 175:91-97.

The present invention also relates to the administration of a vaccinewhich is co-administered with a molecule capable of modulating immuneresponses. Kim et al. (1997) Nature Biotech. 15:641-646, for example,report the enhancement of immune responses produced by DNA immunizationswhen DNA sequences encoding molecules which stimulate the immuneresponse are co-administered. In a similar fashion, the vaccines of thepresent invention may be co-administered with either nucleic acidsencoding immune modulators or the immune modulators themselves. Theseimmune modulators include granulocyte macrophage colony stimulatingfactor (GM-CSF) and CD86.

The vaccines of the present invention may be used to confer resistanceto staphylococcal infection by either passive or active immunization.When the vaccines of the present invention are used to confer resistanceto staphylococcal infection through active immunization, a vaccine ofthe present invention is administered to an animal to elicit aprotective immune response which either prevents or attenuates astaphylococcal infection. When the vaccines of the present invention areused to confer resistance to staphylococcal infection through passiveimmunization, the vaccine is provided to a host animal (e.g., human,dog, or mouse), and the antisera elicited by this antisera is recoveredand directly provided to a recipient suspected of having an infectioncaused by a member of the Staphylococcus genus.

The ability to label antibodies, or fragments of antibodies, with toxinmolecules provides an additional method for treating staphylococcalinfections when passive immunization is conducted. In this embodiment,antibodies, or fragments of antibodies, capable of recognizing the S.aureus polypeptides disclosed herein, or fragments thereof, as well asother Staphylococcus proteins, are labeled with toxin molecules prior totheir administration to the patient. When such toxin derivatizedantibodies bind to Staphylococcus cells, toxin moieties will belocalized to these cells and will cause their death.

The present invention thus concerns and provides a means for preventingor attenuating a staphylococcal infection resulting from organisms whichhave antigens that are recognized and bound by antisera produced inresponse to the polypeptides of the present invention. As used herein, avaccine is said to prevent or attenuate a disease if its administrationto an animal results either in the total or partial attenuation (i.e.,suppression) of a symptom or condition of the disease, or in the totalor partial immunity of the animal to the disease.

The administration of the vaccine (or the antisera which it elicits) maybe for either a “prophylactic” or “therapeutic” purpose. When providedprophylactically, the compound(s) are provided in advance of anysymptoms of staphylococcal infection. The prophylactic administration ofthe compound(s) serves to prevent or attenuate any subsequent infection.When provided therapeutically, the compound(s) is provided upon or afterthe detection of symptoms which indicate that an animal may be infectedwith a member of the Staphylococcus genus. The therapeuticadministration of the compound(s) serves to attenuate any actualinfection. Thus, the S. aureus polypeptides, and fragments thereof, ofthe present invention may be provided either prior to the onset ofinfection (so as to prevent or attenuate an anticipated infection) orafter the initiation of an actual infection.

The polypeptides of the invention, whether encoding a portion of anative protein or a functional derivative thereof, may be administeredin pure form or may be coupled to a macromolecular carrier. Example ofsuch carriers are proteins and carbohydrates. Suitable proteins whichmay act as macromolecular carrier for enhancing the immunogenicity ofthe polypeptides of the present invention include keyhole limpethemacyanin (KLH) tetanus toxoid, pertussis toxin, bovine serum albumin,and ovalbumin. Methods for coupling the polypeptides of the presentinvention to such macromolecular carriers are disclosed in Harlow etal., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988).

A composition is said to be “pharmacologically or physiologicallyacceptable” if its administration can be tolerated by a recipient animaland is otherwise suitable for administration to that animal. Such anagent is said to be administered in a “therapeutically effective amount”if the amount administered is physiologically significant. An agent isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient patient.

While in all instances the vaccine of the present invention isadministered as a pharmacologically acceptable compound, one skilled inthe art would recognize that the composition of a pharmacologicallyacceptable compound varies with the animal to which it is administered.For example, a vaccine intended for human use will generally not beco-administered with Freund's adjuvant. Further, the level of purity ofthe S. aureus polypeptides of the present invention will normally behigher when administered to a human than when administered to anon-human animal.

As would be understood by one of ordinary skill in the art, when thevaccine of the present invention is provided to an animal, it may be ina composition which may contain salts, buffers, adjuvants, or othersubstances which are desirable for improving the efficacy of thecomposition. Adjuvants are substances that can be used to specificallyaugment a specific immune response. These substances generally performtwo functions: (1) they protect the antigen(s) from being rapidlycatabolized after administration and (2) they nonspecifically stimulateimmune responses.

Normally, the adjuvant and the composition are mixed prior topresentation to the immune system, or presented separately, but into thesame site of the animal being immunized. Adjuvants can be looselydivided into several groups based upon their composition. These groupsinclude oil adjuvants (for example, Freund's complete and incomplete),mineral salts (for example, AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄(SO₄), silica,kaolin, and carbon), polynucleotides (for example, poly IC and poly AUacids), and certain natural substances (for example, wax D fromMycobacterium tuberculosis, as well as substances found inCorynebacterium parvum, or Bordetella pertussis, and members of thegenus Brucella. Other substances useful as adjuvants are the saponinssuch as, for example, Quil A. (Superfos A/S, Denmark). Preferredadjuvants for use in the present invention include aluminum salts, suchas AlK(SO₄)₂, AlNa(SO₄)₂, and AlNH₄(SO₄). Examples of materials suitablefor use in vaccine compositions are provided in REMINGTON'SPHARMACEUTICAL SCIENCES 1324-1341 (A. Osol, ed, Mack Publishing Co,Easton, Pa., (1980) (incorporated herein by reference).

The therapeutic compositions of the present invention can beadministered parenterally by injection, rapid infusion, nasopharyngealabsorption (intranasopharangeally), dermoabsorption, or orally. Thecompositions may alternatively be administered intramuscularly, orintravenously. Compositions for parenteral administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Carriers or occlusive dressings can be used toincrease skin permeability and enhance antigen absorption. Liquid dosageforms for oral administration may generally comprise a liposome solutioncontaining the liquid dosage form. Suitable forms for suspendingliposomes include emulsions, suspensions, solutions, syrups, and elixirscontaining inert diluents commonly used in the art, such as purifiedwater. Besides the inert diluents, such compositions can also includeadjuvants, wetting agents, emulsifying and suspending agents, orsweetening, flavoring, or perfuming agents.

Therapeutic compositions of the present invention can also beadministered in encapsulated form. For example, intranasal immunizationusing vaccines encapsulated in biodegradable microsphere composed ofpoly(DL-lactide-co-glycolide). See, Shahin, R. et al. (1995) Infect.Immun. 63:1195-1200. Similarly, orally administered encapsulatedSalmonella typhimurium antigens can also be used. Allaoui-Attarki, K. etal. (1997) Infect. Immun. 65:853-857. Encapsulated vaccines of thepresent invention can be administered by a variety of routes includingthose involving contacting the vaccine with mucous membranes (e.g.,intranasally, intracolonicly, intraduodenally).

Many different techniques exist for the timing of the immunizations whena multiple administration regimen is utilized. It is possible to use thecompositions of the invention more than once to increase the levels anddiversities of expression of the immunoglobulin repertoire expressed bythe immunized animal. Typically, if multiple immunizations are given,they will be given one to two months apart.

According to the present invention, an “effective amount” of atherapeutic composition is one which is sufficient to achieve a desiredbiological effect. Generally, the dosage needed to provide an effectiveamount of the composition will vary depending upon such factors as theanimal's or human's age, condition, sex, and extent of disease, if any,and other variables which can be adjusted by one of ordinary skill inthe art.

The antigenic preparations of the invention can be administered byeither single or multiple dosages of an effective amount. Effectiveamounts of the compositions of the invention can vary from 0.01-1,000μg/ml per dose, more preferably 0.1-500 μg/ml per dose, and mostpreferably 10-300 μg/ml per dose.

EXAMPLES Example 1 Isolation of a Selected DNA Clone from the DepositedSample

Three approaches can be used to isolate a S. aureus clone comprising apolynucleotide of the present invention from any S. aureus genomic DNAlibrary. The S. aureus strain ISP3 has been deposited as a convienentsource for obtaining a S. aureus strain although a wide varity ofstrains S. aureus strains can be used which are known in the art.

S. aureus genomic DNA is prepared using the following method. A 20 mlovernight bacterial culture grown in a rich medium (e.g., Trypticase SoyBroth, Brain Heart Infusion broth or Super broth), pelleted, washed twotimes with TES (30 mM Tris-pH 8.0, 25 mM EDTA, 50 mM NaCl), andresuspended in 5 ml high salt TES (2.5M NaCl). Lysostaphin is added tofinal concentration of approx 50 ug/ml and the mixture is rotated slowly1 hour at 37 C to make protoplast cells. The solution is then placed inincubator (or place in a shaking water bath) and warmed to 55 C. Fivehundred micro liter of 20% sarcosyl in TES (final concentration 2%) isthen added to lyse the cells. Next, guanidine HCl is added to a finalconcentration of 7M (3.69 g in 5.5 ml). The mixture is swirled slowly at55 C for 60-90 min (solution should clear). A CsCl gradient is then setup in SW41 ultra clear tubes using 2.0 ml 5.7M CsCl and overlaying with2.85M CsCl. The gradient is carefully overlayed with the DNA-containingGuHCl solution. The gradient is spun at 30,000 rpm, 20 C for 24 hr andthe lower DNA band is collected. The volume is increased to 5 ml with TEbuffer. The DNA is then treated with protease K (10 ug/ml) overnight at37 C, and precipitated with ethanol. The precipitated DNA is resuspendedin a desired buffer.

In the first method, a plasmid is directly isolated by screening aplasmid S. aureus genomic DNA library using a polynucleotide probecorresponding to a polynucleotide of the present invention.Particularly, a specific polynucleotide with 30-40 nucleotides issynthesized using an Applied Biosystems DNA synthesizer according to thesequence reported. The oligonucleotide is labeled, for instance, with³²P-γ-ATP using T4 polynucleotide kinase and purified according toroutine methods. (See, e.g., Maniatis et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).)The library is transformed into a suitable host, as indicated above(such as XL-1 Blue (Stratagene)) using techniques known to those ofskill in the art. See, e.g., Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL (Cold Spring Harbor, N.Y. 2nd ed. 1989); Ausubel etal., CURRENT PROTOCALS IN MOLECULAR BIOLOGY (John Wiley and Sons, N.Y.1989). The transformants are plated on 1.5% agar plates (containing theappropriate selection agent, e.g., ampicillin) to a density of about 150transformants (colonies) per plate. These plates are screened usingNylon membranes according to routine methods for bacterial colonyscreening. See, e.g., Sambrook et al. MOLECULAR CLONING: A LABORATORYMANUAL (Cold Spring Harbor, N.Y. 2nd ed. 1989); Ausubel et al., CURRENTPROTOCALS IN MOLECULAR BIOLOGY (John Wiley and Sons, N.Y. 1989) or othertechniques known to those of skill in the art.

Alternatively, two primers of 15-25 nucleotides derived from the 5′ and3′ ends of a polynucleotide of Table 1 are synthesized and used toamplify the desired DNA by PCR using a S. aureus genomic DNA prep (e.g.,the deposited S. aureus ISP3) as a template. PCR is carried out underroutine conditions, for instance, in 25 μl of reaction mixture with 0.5ug of the above DNA template. A convenient reaction mixture is 1.5-5 mMMgCl₂, 0.01% (w/v) gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cyclesof PCR (denaturation at 94° C. for 1 min; annealing at 55° C. for 1 min;elongation at 72° C. for 1 min) are performed with a Perkin-Elmer Cetusautomated thermal cycler. The amplified product is analyzed by agarosegel electrophoresis and the DNA band with expected molecular weight isexcised and purified. The PCR product is verified to be the selectedsequence by subcloning and sequencing the DNA product.

Finally, overlapping oligos of the DNA sequences of Table 1 can besynthesized and used to generate a nucleotide sequence of desired lengthusing PCR methods known in the art.

Example 2(a) Expression and Purification Staphylococcal Polypeptides inE. coli

The bacterial expression vector pQE60 is used for bacterial expressionin this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif.,91311). pQE60 encodes ampicillin antibiotic resistance (“Ampr^(r)”) andcontains a bacterial origin of replication (“ori”), an IPTG induciblepromoter, a ribosome binding site (“RBS”), six codons encoding histidineresidues that allow affinity purification usingnickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin (QIAGEN, Inc.,supra) and suitable single restriction enzyme cleavage sites. Theseelements are arranged such that an inserted DNA fragment encoding apolypeptide expresses that polypeptide with the six His residues (i.e.,a “6×His tag”) covalently linked to the carboxyl terminus of thatpolypeptide.

The DNA sequence encoding the desired portion of a S. aureus protein ofthe present invention is amplified from S. aureus genomic DNA or fromthe deposited DNA clone using PCR oligonucleotide primers which annealto the 5′ and 3′ sequences coding for the portion of the S. aureuspolynucleotide. Additional nucleotides containing restriction sites tofacilitate cloning in the pQE60 vector are added to the 5′ and 3′sequences, respectively.

For cloning the mature protein, the 5′ primer has a sequence containingan appropriate restriction site followed by nucleotides of the aminoterminal coding sequence of the desired S. aureus polynucleotidesequence in Table 1. One of ordinary skill in the art would appreciatethat the point in the protein coding sequence where the 5′ and 3′primers begin may be varied to amplify a DNA segment encoding anydesired portion of the complete protein shorter or longer than themature form. The 3′ primer has a sequence containing an appropriaterestriction site followed by nucleotides complementary to the 3′ end ofthe desired coding sequence of Table 1, excluding a stop codon, with thecoding sequence aligned with the restriction site so as to maintain itsreading frame with that of the six His codons in the pQE60 vector.

The amplified S. aureus DNA fragment and the vector pQE60 are digestedwith restriction enzymes which recognize the sites in the primers andthe digested DNAs are then ligated together. The S. aureus DNA isinserted into the restricted pQE60 vector in a manner which places theS. aureus protein coding region downstream from the IPTG-induciblepromoter and in-frame with an initiating AUG and the six histidinecodons.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures such as those described by Sambrook et al., supra.E. coli strain M15/rep4, containing multiple copies of the plasmidpREP4, which expresses the lac repressor and confers kanamycinresistance (“Kan^(r)”), is used in carrying out the illustrative exampledescribed herein. This strain, which is only one of many that aresuitable for expressing a S. aureus polypeptide, is availablecommercially (QIAGEN, Inc., supra). Transformants are identified bytheir ability to grow on LB plates in the presence of ampicillin andkanamycin. Plasmid DNA is isolated from resistant colonies and theidentity of the cloned DNA confirmed by restriction analysis, PCR andDNA sequencing.

Clones containing the desired constructs are grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml). The O/N culture is used to inoculate a largeculture, at a dilution of approximately 1:25 to 1:250. The cells aregrown to an optical density at 600 nm (“OD⁶⁰⁰”) of between 0.4 and 0.6.Isopropyl-β-D-thiogalactopyranoside .(“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from the lac repressorsensitive promoter, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation.

The cells are then stirred for 3-4 hours at 4° C. in 6M guanidine-HCl,pH 8. The cell debris is removed by centrifugation, and the supernatantcontaining the S. aureus polypeptide is loaded onto anickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column (QIAGEN,Inc., supra). Proteins with a 6×His tag bind to the Ni-NTA resin withhigh affinity and can be purified in a simple one-step procedure (fordetails see: The QLAexpressionist, 1995, QIAGEN, Inc., supra). Brieflythe supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8,the column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8,then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally theS. aureus polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins can be eluted by the addition of 250 mMimidazole. Imidazole is removed by a final dialyzing step against PBS or50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified proteinis stored at 4° C. or frozen at −80° C.

Alternatively, the polypeptides of the present invention can be producedby a non-denaturing method. In this method, after the cells areharvested by centrifugation, the cell pellet from each liter of cultureis resuspended in 25 ml of Lysis Buffer A at 4° C. (Lysis Buffer A=50 mMNa-phosphate, 300 mM NaCl, 10 mM 2-mercaptoethanol, 10% Glycerol, pH 7.5with 1 tablet of Complete EDTA-free protease inhibitor cocktail(Boehringer Mannheim #1873580) per 50 ml of buffer). Absorbance at 550nm is approximately 10-20 O.D./ml. The suspension is then put throughthree freeze/thaw cycles from −70° C. (using a ethanol-dry ice bath) upto room temperature. The cells are lysed via sonication in short 10 secbursts over 3 minutes at approximately 80W while kept on ice. Thesonicated sample is then centrifuged at 15,000 RPM for 30 minutes at 4°C. The supernatant is passed through a column containing 1.0 ml of CL-4Bresin to pre-clear the sample of any proteins that may bind to agarosenon-specifically, and the flow-through fraction is collected.

The pre-cleared flow-through is applied to a nickel-nitrilo-tri-aceticacid (“Ni-NTA”) affinity resin column (Qiagen, Inc., supra). Proteinswith a 6×His tag bind to the Ni-NTA resin with high affinity and can bepurified in a simple one-step procedure. Briefly, the supernatant isloaded onto the column in Lysis Buffer A at 4° C., the column is firstwashed with 10 volumes of Lysis Buffer A until the A280 of the eluatereturns to the baseline. Then, the column is washed with 5 volumes of 40mM Imidazole (92% Lysis Buffer A/8% Buffer B) (Buffer B=50 mMNa-Phosphate, 300 mM NaCl, 10% Glycerol, 10 mM 2-mercaptoethanol, 500 mMImidazole, pH of the final buffer should be 7.5). The protein is elutedoff of the column with a series of increasing Imidazole solutions madeby adjusting the ratios of Lysis Buffer A to Buffer B. Three differentconcentrations are used: 3 volumes of 75 mM Imidazole, 3 volumes of 150mM Imidazole, 5 volumes of 500 mM Imidazole. The fractions containingthe purified protein are analyzed using 8%, 10% or 14% SDS-PAGEdepending on the protein size. The purified protein is then dialyzed 2×against phosphate-buffered saline (PBS) in order to place it into aneasily workable buffer. The purified protein is stored at 4° C. orfrozen at −80°

The following is another alternative method may be used to purify S.aureus expressed in E coli when it is present in the form of inclusionbodies. Unless otherwise specified, all of the following steps areconducted at 4-10° C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10° C. and the cells are harvested bycontinuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basisof the expected yield of protein per unit weight of cell paste and theamount of purified protein required, an appropriate amount of cellpaste, by weight, is suspended in a buffer solution containing 100 mMTris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneoussuspension using a high shear mixer.

The cells are then lysed by passing the solution through amicrofluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the S. aureuspolypeptide-containing supernatant is incubated at 4° C. overnight toallow further GuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4° C. without mixing for 12 hours prior tofurther purification steps.

To clarify the refolded S. aureus polypeptide solution, a previouslyprepared tangential filtration unit equipped with 0.16 μm membranefilter with appropriate surface area (e.g., Filtron), equilibrated with40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loadedonto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems).The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in astepwise manner. The absorbance at 280 mm of the effluent iscontinuously monitored. Fractions are collected and further analyzed bySDS-PAGE.

Fractions containing the S. aureus polypeptide are then pooled and mixedwith 4 volumes of water. The diluted sample is then loaded onto apreviously prepared set of tandem columns of strong anion (Poros HQ-50,Perseptive Biosystems) and weak anion (Poros CM-20, PerseptiveBiosystems) exchange resins. The columns are equilibrated with 40 mMsodium acetate, pH 6.0. Both columns are washed with 40 mM sodiumacetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodiumacetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractionsare collected under constant A₂₈₀ monitoring of the effluent. Fractionscontaining the S. aureus polypeptide (determined, for instance, by 16%SDS-PAGE) are then pooled.

The resultant S. aureus polypeptide exhibits greater than 95% purityafter the above refolding and purification steps. No major contaminantbands are observed from Commassie blue stained 16% SDS-PAGE gel when 5μg of purified protein is loaded. The purified protein is also testedfor endotoxin/LPS contamination, and typically the LPS content is lessthan 0.1 ng/ml according to LAL assays.

Example 2(b) Expression and Purification Staphylococcal Polypeptides inE. coli

Alternatively, the vector pQE10 can be used to clone and expresspolypeptides of the present invention. The difference being such that aninserted DNA fragment encoding a polypeptide expresses that polypeptidewith the six His residues (i.e., a “6×His tag”) covalently linked to theamino terminus of that polypeptide. The bacterial expression vectorpQE10 (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311) isused in this example. The components of the pQE10 plasmid are arrangedsuch that the inserted DNA sequence encoding a polypeptide of thepresent invention expresses the polypeptide with the six His residues(i.e., a “6×His tag”)) covalently linked to the amino terminus.

The DNA sequences encoding the desired portions of a polypeptide ofTable 1 or expressed by the plasmids listed in Table 1 are amplifiedusing PCR oligonucleotide primers from either genomic S. aureus DNA orDNA from the plasmid clones listed in Table 1 clones of the presentinvention. The PCR primers anneal to the nucleotide sequences encodingthe desired amino acid sequence of a polypeptide of the presentinvention. Additional nucleotides containing restriction sites tofacilitate cloning in the pQE10 vector are added to the 5′ and 3′ primersequences, respectively.

For cloning a polypeptide of the present invention, the 5′ and 3′primers are selected to amplify their respective nucleotide codingsequences. One of ordinary skill in the art would appreciate that thepoint in the protein coding sequence where the 5′ and 3′ primers beginsmay be varied to amplify a DNA segment encoding any desired portion of apolypeptide of the present invention. The 5′ primer is designed so thecoding sequence of the 6×His tag is aligned with the restriction site soas to maintain its reading frame with that of S. aureus polypeptide. The3′ is designed to include an stop codon. The amplified DNA fragment isthen cloned, and the protein expressed, as described above for the pQE60plasmid.

The DNA sequences encoding the amino acid sequences of Table 1 may alsobe cloned and expressed as fusion proteins by a protocol similar to thatdescribed directly above, wherein the pET-32b(+) vector (Novagen, 601Science Drive, Madison, Wis. 53711) is preferentially used in place ofpQE10.

Example 2(c) Expression and Purification of Stahphlococcusl Polypeptidesin E. coli

The bacterial expression vector pQE60 is used for bacterial expressionin this example (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif.,91311). However, in this example, the polypeptide coding sequence isinserted such that translation of the six His codons is prevented and,therefore, the polypeptide is produced with no 6×His tag.

The DNA sequence encoding the desired portion of the S. aureus aminoacid sequence is amplified from a S. aureus genomic DNA prep using PCRoligonucleotide primers which anneal to the 5′ and 3′ nucleotidesequences corresponding to the desired portion of the S. aureuspolypeptides. Additional nucleotides containing restriction sites tofacilitate cloning in the pQE60 vector are added to the 5′ and 3′ primersequences.

For cloning a S. aureus polypeptides of the present invention, 5′ and 3′primers are selected to amplify their respective nucleotide codingsequences. One of ordinary skill in the art would appreciate that thepoint in the protein coding sequence where the 5′ and 3′ primers beginmay be varied to amplify a DNA segment encoding any desired portion of apolypeptide of the present invention. The 3′ and 5′ primers containappropriate restriction sites followed by nucleotides complementary tothe 5′ and 3′ ends of the coding sequence respectively. The 3′ primer isadditionally designed to include an in-frame stop codon.

The amplified S. aureus DNA fragments and the vector pQE60 are digestedwith restriction enzymes recognizing the sites in the primers and thedigested DNAs are then ligated together. Insertion of the S. aureus DNAinto the restricted pQE60 vector places the S. aureus protein codingregion including its associated stop codon downstream from theIPTG-inducible promoter and in-frame with an initiating AUG. Theassociated stop codon prevents translation of the six histidine codonsdownstream of the insertion point.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures such as those described by Sambrook et al. E. colistrain M15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses the lac repressor and confers kanamycin resistance(“Kan^(r)”), is used in carrying out the illustrative example describedherein. This strain, which is only one of many that are suitable forexpressing S. aureus polypeptide, is available commercially (QIAGEN,Inc., supra). Transformants are identified by their ability to grow onLB plates in the presence of ampicillin and kanamycin. Plasmid DNA isisolated from resistant colonies and the identity of the cloned DNAconfirmed by restriction analysis, PCR and DNA sequencing.

Clones containing the desired constructs are grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml). The O/N culture is used to inoculate a largeculture, at a dilution of approximately 1:25 to 1:250. The cells aregrown to an optical density at 600 nm (“OD600”) of between 0.4 and 0.6.isopropyl-b-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from the lac repressorsensitive promoter, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation.

To purify the S. aureus polypeptide, the cells are then stirred for 3-4hours at 4° C. in 6M guanidine-HCl, pH 8. The cell debris is removed bycentrifugation, and the supernatant containing the S. aureus polypeptideis dialyzed against 50 mM Na-acetate buffer pH 6, supplemented with 200mM NaCl. Alternatively, the protein can be successfully refolded bydialyzing it against 500 mM NaCl, 20% glycerol, 25 mM Tris/HCl pH 7.4,containing protease inhibitors. After renaturation the protein can bepurified by ion exchange, hydrophobic interaction and size exclusionchromatography. Alternatively, an affinity chromatography step such asan antibody column can be used to obtain pure S. aureus polypeptide. Thepurified protein is stored at 4° C. or frozen at −80° C.

The following alternative method may be used to purify S. aureuspolypeptides expressed in E coli when it is present in the form ofinclusion bodies. Unless otherwise specified, all of the following stepsare conducted at 4-10° C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10° C. and the cells are harvested bycontinuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basisof the expected yield of protein per unit weight of cell paste and theamount of purified protein required, an appropriate amount of cellpaste, by weight, is suspended in a buffer solution containing 100 mMTris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneoussuspension using a high shear mixer.

The cells ware then lysed by passing the solution through amicrofluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the S. aureuspolypeptide-containing supernatant is incubated at 4° C. overnight toallow further GuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4° C. without mixing for 12 hours prior tofurther purification steps.

To clarify the refolded S. aureus polypeptide solution, a previouslyprepared tangential filtration unit equipped with 0.16 μm membranefilter with appropriate surface area (e.g., Filtron), equilibrated with40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loadedonto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems).The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in astepwise manner. The absorbance at 280 mm of the effluent iscontinuously monitored. Fractions are collected and further analyzed bySDS-PAGE.

Fractions containing the S. aureus polypeptide are then pooled and mixedwith 4 volumes of water. The diluted sample is then loaded onto apreviously prepared set of tandem columns of strong anion (Poros HQ-50,Perseptive Biosystems) and weak anion (Poros CM-20, PerseptiveBiosystems) exchange resins. The columns are equilibrated with 40 mMsodium acetate, pH 6.0. Both columns are washed with 40 mM sodiumacetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodiumacetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractionsare collected under constant A₂₈₀ monitoring of the effluent. Fractionscontaining the S. aureus polypeptide (determined, for instance, by 16%SDS-PAGE) are then pooled.

The resultant S. aureus polypeptide exhibits greater than 95% purityafter the above refolding and purification steps. No major contaminantbands are observed from Commassie blue stained 16% SDS-PAGE gel when 5μg of purified protein is loaded. The purified protein is also testedfor endotoxin/LPS contamination, and typically the LPS content is lessthan 0.1 ng/ml according to LAL assays.

Example 2(d) Cloning and Expression of S. aureus in Other Bacteria

S. aureus polypeptides can also be produced in: S. aureus using themethods of S. Skinner et al., (1988) Mol. Microbiol. 2:289-297 or J. I.Moreno (1996) Protein Expr. Purif. 8(3):332-340; Lactobacillus using themethods of C. Rush et al., 1997 Appl. Microbiol. Biotechnol.47(5):537-542; or in Bacillus subtilis using the methods Chang et al.,U.S. Pat. No. 4,952,508.

Example 3 Cloning and Expression in COS Cells

A S. aureus expression plasmid is made by cloning a portion of the DNAencoding a S. aureus polypeptide into the expression vector pDNAI/Amp orpDNAIII (which can be obtained from Invitrogen, Inc.). The expressionvector pDNAI/amp contains: (1) an E. coli origin of replicationeffective for propagation in E. coli and other prokaryotic cells; (2) anampicillin resistance gene for selection of plasmid-containingprokaryotic cells; (3) an SV40 origin of replication for propagation ineukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron; (5)several codons encoding a hemagglutinin fragment (i.e., an “HA” tag tofacilitate purification) followed by a termination codon andpolyadenylation signal arranged so that a DNA can be conveniently placedunder expression control of the CMV promoter and operably linked to theSV40 intron and the polyadenylation signal by means of restriction sitesin the polylinker. The HA tag corresponds to an epitope derived from theinfluenza hemagglutinin protein described by Wilson et al. 1984 Cell37:767. The fusion of the HA tag to the target protein allows easydetection and recovery of the recombinant protein with an antibody thatrecognizes the HA epitope. pDNAIII contains, in addition, the selectableneomycin marker.

A DNA fragment encoding a S. aureus polypeptide is cloned into thepolylinker region of the vector so that recombinant protein expressionis directed by the CMV promoter. The plasmid construction strategy is asfollows. The DNA from a S. aureus genomic DNA prep is amplified usingprimers that contain convenient restriction sites, much as describedabove for construction of vectors for expression of S. aureus in E.coli. The 5′ primer contains a Kozak sequence, an AUG start codon, andnucleotides of the 5′ coding region of the S. aureus polypeptide. The 3′primer, contains nucleotides complementary to the 3′ coding sequence ofthe S. aureus DNA, a stop codon, and a convenient restriction site.

The PCR amplified DNA fragment and the vector, pDNAI/Amp, are digestedwith appropriate restriction enzymes and then ligated. The ligationmixture is transformed into an appropriate E. coli strain such as SURE™(Stratagene Cloning Systems, La Jolla, Calif. 92037), and thetransformed culture is plated on ampicillin media plates which then areincubated to allow growth of ampicillin resistant colonies. Plasmid DNAis isolated from resistant colonies and examined by restriction analysisor other means for the presence of the fragment encoding the S. aureuspolypeptide

For expression of a recombinant S. aureus polypeptide, COS cells aretransfected with an expression vector, as described above, usingDEAE-dextran, as described, for instance, by Sambrook et al. (supra).Cells are incubated under conditions for expression of S. aureus by thevector.

Expression of the S. aureus-HA fusion protein is detected byradiolabeling and immunoprecipitation, using methods described in, forexample Harlow et al., supra. To this end, two days after transfection,the cells are labeled by incubation in media containing ³⁵S-cysteine for8 hours. The cells and the media are collected, and the cells are washedand the lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1%NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described byWilson et al. (supra). Proteins are precipitated from the cell lysateand from the culture media using an HA-specific monoclonal antibody. Theprecipitated proteins then are analyzed by SDS-PAGE and autoradiography.An expression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 4 Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of S. aureus polypeptide inthis example. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCCAccession No. 37146). The plasmid contains the mouse DHFR gene undercontrol of the SV40 early promoter. Chinese hamster ovary cells or othercells lacking dihydrofolate activity that are transfected with theseplasmids can be selected by growing the cells in a selective medium(alpha minus MEM, Life Technologies) supplemented with thechemotherapeutic agent methotrexate. The amplification of the DHFR genesin cells resistant to methotrexate (MTX) has been well documented. See,e.g., Alt et al., 1978, J. Biol. Chem. 253:1357-1370; Hamlin et al.,1990, Biochem. et Biophys. Acta, 1097:107-143; Page et al., 1991,Biotechnology 9:64-68. Cells grown in increasing concentrations of MTXdevelop resistance to the drug by overproducing the target enzyme, DHFR,as a result of amplification of the DHFR gene. If a second gene islinked to the DHFR gene, it is usually co-amplified and over-expressed.It is known in the art that this approach may be used to develop celllines carrying more than 1,000 copies of the amplified gene(s).Subsequently, when the methotrexate is withdrawn, cell lines areobtained which contain the amplified gene integrated into one or morechromosome(s) of the host cell.

Plasmid pC4 contains the strong promoter of the long terminal repeat(LTR) of the Rouse Sarcoma Virus, for expressing a polypeptide ofinterest, Cullen, et al. (1985) Mol. Cell. Biol. 5:438-447; plus afragment isolated from the enhancer of the immediate early gene of humancytomegalovirus (CMV), Boshart, et al., 1985, Cell 41:521-530.Downstream of the promoter are the following single restriction enzymecleavage sites that allow the integration of the genes: Bam HI, Xba I,and Asp 718. Behind these cloning sites the plasmid contains the 3′intron and polyadenylation site of the rat preproinsulin gene. Otherhigh efficiency promoters can also be used for the expression, e.g., thehuman β-actin promoter, the SV40 early or late promoters or the longterminal repeats from other retroviruses, e.g., HIV and HTLVI.Clontech's Tet-Off and Tet-On gene expression systems and similarsystems can be used to express the S. aureus polypeptide in a regulatedway in mammalian cells (Gossen et al., 1992, Proc. Natl. Acad. Sci. USA89:5547-5551. For the polyadenylation of the mRNA other signals, e.g.,from the human growth hormone or globin genes can be used as well.Stable cell lines carrying a gene of interest integrated into thechromosomes can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g., G418 plusmethotrexate.

The plasmid pC4 is digested with the restriction enzymes and thendephosphorylated using calf intestinal phosphates by procedures known inthe art. The vector is then isolated from a 1% agarose gel. The DNAsequence encoding the S. aureus polypeptide is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thedesired portion of the gene. A 5′ primer containing a restriction site,a Kozak sequence, an AUG start codon, and nucleotides of the 5′ codingregion of the S. aureus polypeptide is synthesized and used. A 3′primer, containing a restriction site, stop codon, and nucleotidescomplementary to the 3′ coding sequence of the S. aureus polypeptides issynthesized and used. The amplified fragment is digested with therestriction endonucleases and then purified again on a 1% agarose gel.The isolated fragment and the dephosphorylated vector are then ligatedwith T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are thentransformed and bacteria are identified that contain the fragmentinserted into plasmid pC4 using, for instance, restriction enzymeanalysis.

Chinese hamster ovary cells lacking an active DHFR gene are used fortransfection. Five μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSVneo using a lipid-mediated transfectionagent such as Lipofectin™ or LipofectAMINE.™ (LifeTechnologiesGaithersburg, Md.). The plasmid pSV2-neo contains a dominant selectablemarker, the neo gene from Tn5 encoding an enzyme that confers resistanceto a group of antibiotics including G418. The cells are seeded in alphaminus MEM supplemented with 1 mg/ml G418. After 2 days, the cells aretrypsinized and seeded in hybridoma cloning plates (Greiner, Germany) inalpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexateplus 1 mg/ml G418. After about 10-14 days single clones are trypsinizedand then seeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 5 Quantitative Murine Soft Tissue Infection Model for S. aureus

Compositions of the present invention, including polypeptides andpeptides, are assayed for their ability to function as vaccines or toenhance/stimulate an immune response to a bacterial species (e.g., S.aureus) using the following quantitative murine soft tissue infectionmodel. Mice (e.g., NIH Swiss female mice, approximately 7 weeks old) arefirst treated with a biologically protective effective amount, or immuneenhancing/stimulating effective amount of a composition of the presentinvention using methods known in the art, such as those discussed above.See, e.g., Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988). An example of an appropriatestarting dose is 20 ug per animal.

The desired bacterial species used to challenge the mice, such as S.aureus, is grown as an overnight culture. The culture is diluted to aconcentration of 5×10⁸ cfu/ml, in an appropriate media, mixed well,serially diluted, and titered. The desired doses are further diluted 1:2with sterilized Cytodex 3 microcarrier beads preswollen in sterile PBS(3 g/100 ml). Mice are anesthetize briefly until docile, but stillmobile and injected with 0.2 ml of the Cytodex 3 bead/bacterial mixtureinto each animal subcutaneously in the inguinal region. After four days,counting the day of injection as day one, mice are sacrificed and thecontents of the abscess is excised and placed in a 15 ml conical tubecontaining 1.0 ml of sterile PBS. The contents of the abscess is thenenzymatically treated and plated as follows.

The abscess is first disrupted by vortexing with sterilized glass beadsplaced in the tubes. 3.0 mls of prepared enzyme mixture (1.0 mlCollagenase D (4.0 mg/ml), 1.0 ml Trypsin (6.0 mg/ml) and 8.0 ml PBS) isthen added to each tube followed by a 20 min. incubation at 37 C. Thesolution is then centrifuged and the supernatant drawn off. 0.5 ml dH20is then added and the tubes are vortexed and then incubated for 10 min.at room temperature. 0.5 ml media is then added and samples are seriallydiluted and plated onto agar plates, and grown overnight at 37 C. Plateswith distinct and separate colonies are then counted, compared topositive and negative control samples, and quantified. The method can beused to identify composition and determine appropriate and effectivedoses for humans and other animals by comparing the effective doses ofcompositions of the present invention with compositions known in the artto be effective in both mice and humans. Doses for the effectivetreatment of humans and other animals, using compositions of the presentinvention, are extrapolated using the data from the above experiments ofmice. It is appreciated that further studies in humans and other animalsmay be needed to determine the most effective doses using methods ofclinical practice known in the art.

Example 6 Murine Systemic Neutropenic Model for S. aureus Infection

Compositions of the present invention, including polypeptides andpeptides, are assayed for their ability to function as vaccines or toenhance/stimulate an immune response to a bacterial species (e.g., S.aureus) using the following qualitative murine systemic neutropenicmodel. Mice (e.g., NIH Swiss female mice, approximately 7 weeks old) arefirst treated with a biologically protective effective amount, or immuneenhancing/stimulating effective amount of a composition of the presentinvention using methods known in the art, such as those discussed above.See, e.g., Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988). An example of an appropriatestarting dose is 20 ug per animal.

Mice are then injected with 250-300 mg/kg cyclophosphamideintraperitonially. Counting the day of C.P. injection as day one, themice are left untreated for 5 days to begin recovery of PMNL'S.

The desired bacterial species used to challenge the mice, such as S.aureus, is grown as an overnight culture. The culture is diluted to aconcentration of 5×10⁸ cfu/ml, in an appropriate media, mixed well,serially diluted, and titered. The desired doses are further diluted 1:2in 4% Brewer's yeast in media.

Mice are injected with the bacteria/brewer's yeast challengeintraperitonially. The Brewer's yeast solution alone is used as acontrol. The mice are then monitored twice daily for the first weekfollowing challenge, and once a day for the next week to ascertainmorbidity and mortality. Mice remaining at the end of the experiment aresacrificed. The method can be used to identify compositions anddetermine appropriate and effective doses for humans and other animalsby comparing the effective doses of compositions of the presentinvention with compositions known in the art to be effective in bothmice and humans. Doses for the effective treatment of humans and otheranimals, using compositions of the present invention, are extrapolatedusing the data from the above experiments of mice. It is appreciatedthat further studies in humans and other animals may be needed todetermine the most effective doses using methods of clinical practiceknown in the art.

The disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference in theirentireties.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention. Functionally equivalent methodsand components are within the scope of the invention, in addition tothose shown and described herein and will become apparent to thoseskilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 22 <210> SEQ ID NO 1 <211> LENGTH: 1470<212> TYPE: DNA <213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 1tggaaaatta atgaagttcc aaagtttaga tcaaaactgg aataatggtg ga#tggcgtaa     60agcagaggtt gcacataaag ttgttcataa ttatgaaaat gatatgattt tt#attagacc    120atttaaaaaa gcataattta aatcgaaggc aggacattga aatatgaaat tt#tcaacttt    180aagtgaagaa gaatttacca actacaccaa aaagcacttc aaacattata cg#cagtctat    240agaattatat aattatagaa ataaaataaa tcatgaagca catattgtgg ga#gtgaagaa    300tgataaaaat gaagttatag ctgcatgttt attaacagag gcacgaattt tt#aaattcta    360caaatatttc tactctcata gaggtccttt acttgattat ttcgatgcta aa#ttagtttg    420ttactttttt aaagaattat ctaaattcat ttataaaaat agaggagtat tt#attcttgt    480tgatccatat ttaatagaga atttaagaga tgcaaatggt aggataataa ag#aattataa    540taattcagtg atagtaaaga tgctagggaa aattgggtat ctccatcaag gt#tatacaac    600aggatattca aataaaagtc aaattaggtg gatttctgta ttggatttaa aa#gataaaga    660tgagaatcaa cttttaaaag aaatggaata ccaaactaga agaaatataa aa#aagactat    720tgagattggt gttaaggttg aagatttatc tattgaagaa acaaatcgat tt#tataaatt    780gtttcaaatg gctgaagaaa aacatggttt tcatttcatg aatgaagatt at#tttaaacg    840aatgcaagaa atatataaag ataaggcaat gttaaagata gcttgtataa at#cttaatga    900atatcaagat aaattaaaaa tacaattatt gaaaatcgaa aatgaaatga tg#actgtgaa    960cagagcatta aatgaaaatc caaattctaa aaaaaataaa tcaaaattaa at#cagttaaa   1020tatgcaatta tctagtatta ataatagaat tagtaaaacc gaagaactaa ta#tttgaaga   1080tggacctgtt ttggatttag ctgctgcttt atttatatgt actgatgatg aa#gtttatta   1140tctatcaagt ggatcaaatc cgaaatataa tcagtatatg ggtgcatatc at#ctacaatg   1200gcatatgata aaatatgcaa aatcacataa tattaatagg tataattttt at#ggaataac   1260aggcgtcttt agtaatgagg cggatgattt tggtgttcaa caatttaaaa ag#ggttttaa   1320tgcacatgtt gaagaattaa ttggtgattt catcaaacca gtaagaccaa tt#ctatataa   1380atttgcaaaa cttatttata aggtttaatt ataaagtatg ttggaaattg aa#attttaaa   1440 ttctttccaa catacttttc actttttaag         #                   #         1470 <210> SEQ ID NO 2 <211> LENGTH: 414<212> TYPE: PRT <213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 2Met Lys Phe Ser Thr Leu Ser Glu Glu Glu Ph #e Thr Asn Tyr Thr Lys  1               5  #                 10  #                 15Lys His Phe Lys His Tyr Thr Gln Ser Ile Gl #u Leu Tyr Asn Tyr Arg             20      #             25      #             30Asn Lys Ile Asn His Glu Ala His Ile Val Gl #y Val Lys Asn Asp Lys         35          #         40          #         45Asn Glu Val Ile Ala Ala Cys Leu Leu Thr Gl #u Ala Arg Ile Phe Lys     50              #     55              #     60Phe Tyr Lys Tyr Phe Tyr Ser His Arg Gly Pr #o Leu Leu Asp Tyr Phe 65                  # 70                  # 75                  # 80Asp Ala Lys Leu Val Cys Tyr Phe Phe Lys Gl #u Leu Ser Lys Phe Ile                 85  #                 90  #                 95Tyr Lys Asn Arg Gly Val Phe Ile Leu Val As #p Pro Tyr Leu Ile Glu            100       #           105       #           110Asn Leu Arg Asp Ala Asn Gly Arg Ile Ile Ly #s Asn Tyr Asn Asn Ser        115           #       120           #       125Val Ile Val Lys Met Leu Gly Lys Ile Gly Ty #r Leu His Gln Gly Tyr    130               #   135               #   140Thr Thr Gly Tyr Ser Asn Lys Ser Gln Ile Ar #g Trp Ile Ser Val Leu145                 1 #50                 1 #55                 1 #60Asp Leu Lys Asp Lys Asp Glu Asn Gln Leu Le #u Lys Glu Met Glu Tyr                165   #               170   #               175Gln Thr Arg Arg Asn Ile Lys Lys Thr Ile Gl #u Ile Gly Val Lys Val            180       #           185       #           190Glu Asp Leu Ser Ile Glu Glu Thr Asn Arg Ph #e Tyr Lys Leu Phe Gln        195           #       200           #       205Met Ala Glu Glu Lys His Gly Phe His Phe Me #t Asn Glu Asp Tyr Phe    210               #   215               #   220Lys Arg Met Gln Glu Ile Tyr Lys Asp Lys Al #a Met Leu Lys Ile Ala225                 2 #30                 2 #35                 2 #40Cys Ile Asn Leu Asn Glu Tyr Gln Asp Lys Le #u Lys Ile Gln Leu Leu                245   #               250   #               255Lys Ile Glu Asn Glu Met Met Thr Val Asn Ar #g Ala Leu Asn Glu Asn            260       #           265       #           270Pro Asn Ser Lys Lys Asn Lys Ser Lys Leu As #n Gln Leu Asn Met Gln        275           #       280           #       285Leu Ser Ser Ile Asn Asn Arg Ile Ser Lys Th #r Glu Glu Leu Ile Phe    290               #   295               #   300Glu Asp Gly Pro Val Leu Asp Leu Ala Ala Al #a Leu Phe Ile Cys Thr305                 3 #10                 3 #15                 3 #20Asp Asp Glu Val Tyr Tyr Leu Ser Ser Gly Se #r Asn Pro Lys Tyr Asn                325   #               330   #               335Gln Tyr Met Gly Ala Tyr His Leu Gln Trp Hi #s Met Ile Lys Tyr Ala            340       #           345       #           350Lys Ser His Asn Ile Asn Arg Tyr Asn Phe Ty #r Gly Ile Thr Gly Val        355           #       360           #       365Phe Ser Asn Glu Ala Asp Asp Phe Gly Val Gl #n Gln Phe Lys Lys Gly    370               #   375               #   380Phe Asn Ala His Val Glu Glu Leu Ile Gly As #p Phe Ile Lys Pro Val385                 3 #90                 3 #95                 4 #00Arg Pro Ile Leu Tyr Lys Phe Ala Lys Leu Il #e Tyr Lys Val                405   #               410 <210> SEQ ID NO 3<211> LENGTH: 561 <212> TYPE: DNA <213> ORGANISM: Staphylococcus aureus<400> SEQUENCE: 3tctccgggtg gtgtaattgt agttctactt gttattttac ttatgattac aa#tggcttat     60cagaaaatgc gaatgaagtt taaaaaggga gctaatatca atgaatacaa at#gatgctat    120taaaatttta aaagagaacg gtttaaaata tacagataaa cgtaaagata tg#ttagatat    180ttttgtcgaa gaagataagt atataaacgc aaagtatata caacaagtta tg#gatgaaaa    240ttatcctgga atttcattcg acacaatata tagaaacctg cacttattta aa#gatttagg    300aattattgaa aatacagaac ttgatggtga aatgaagttt agaatcgctt gt#acaaacca    360tcatcatcat cattttatct gtgaaaagtg tggagataca aaggtaatag at#tattgtcc    420aatagatcag ataaaattat cactacctgg tgttaatatt cacaaacaca aa#cttgaagt    480ttatggtgta tgtgagtctt gccaagatta atataaagaa atgagattta tg#cacatttg    540 gtccgatgta tgcataaatc t            #                  #                 561 <210> SEQ ID NO 4 <211> LENGTH: 136<212> TYPE: PRT <213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 4Met Asn Thr Asn Asp Ala Ile Lys Ile Leu Ly #s Glu Asn Gly Leu Lys  1               5  #                 10  #                 15Tyr Thr Asp Lys Arg Lys Asp Met Leu Asp Il #e Phe Val Glu Glu Asp             20      #             25      #             30Lys Tyr Ile Asn Ala Lys Tyr Ile Gln Gln Va #l Met Asp Glu Asn Tyr         35          #         40          #         45Pro Gly Ile Ser Phe Asp Thr Ile Tyr Arg As #n Leu His Leu Phe Lys     50              #     55              #     60Asp Leu Gly Ile Ile Glu Asn Thr Glu Leu As #p Gly Glu Met Lys Phe 65                  # 70                  # 75                  # 80Arg Ile Ala Cys Thr Asn His His His His Hi #s Phe Ile Cys Glu Lys                 85  #                 90  #                 95Cys Gly Asp Thr Lys Val Ile Asp Tyr Cys Pr #o Ile Asp Gln Ile Lys            100       #           105       #           110Leu Ser Leu Pro Gly Val Asn Ile His Lys Hi #s Lys Leu Glu Val Tyr        115           #       120           #       125Gly Val Cys Glu Ser Cys Gln Asp     130               #   135<210> SEQ ID NO 5 <211> LENGTH: 586 <212> TYPE: DNA<213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 5ttaaatgaaa tcatcatgta aatattgaca cgcgcgcaat actacagtta ta#tttatagt     60aagtaataat aattattata taagaaagat ggtgatatag atgagtgttg aa#atagaatc    120aattgaacat gaactagaag aatcaattgc atcattgcga caagcaggcg ta#agaattac    180acctcaaaga caagcaatat tacgttattt aatttcttca catactcatc ca#acagctga    240tgaaatttat caagcacttt cacctgattt tccaaatata agtgttgcga ca#atatataa    300taacttaaga gtgtttaaag atattggaat tgtaaaagaa ttaacatatg ga#gactcatc    360aagtcgattc gactttaata cacataatca ttatcatatt atatgtgaac aa#tgtggtaa    420gattgttgat tttcaatatc cacagttaaa tgaaattgaa agattagctc ag#catatgac    480tgactttgac gtaacacatc atcgaatgga aatttatgga gtttgtaaag aa#tgccaaga    540 taaataattt aactttggta gtatgacaaa ttaaaaaagc gttact   #                586 <210> SEQ ID NO 6 <211> LENGTH: 148 <212> TYPE: PRT<213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 6Met Ser Val Glu Ile Glu Ser Ile Glu His Gl #u Leu Glu Glu Ser Ile  1               5  #                 10  #                 15Ala Ser Leu Arg Gln Ala Gly Val Arg Ile Th #r Pro Gln Arg Gln Ala             20      #             25      #             30Ile Leu Arg Tyr Leu Ile Ser Ser His Thr Hi #s Pro Thr Ala Asp Glu         35          #         40          #         45Ile Tyr Gln Ala Leu Ser Pro Asp Phe Pro As #n Ile Ser Val Ala Thr     50              #     55              #     60Ile Tyr Asn Asn Leu Arg Val Phe Lys Asp Il #e Gly Ile Val Lys Glu 65                  # 70                  # 75                  # 80Leu Thr Tyr Gly Asp Ser Ser Ser Arg Phe As #p Phe Asn Thr His Asn                 85  #                 90  #                 95His Tyr His Ile Ile Cys Glu Gln Cys Gly Ly #s Ile Val Asp Phe Gln            100       #           105       #           110Tyr Pro Gln Leu Asn Glu Ile Glu Arg Leu Al #a Gln His Met Thr Asp        115           #       120           #       125Phe Asp Val Thr His His Arg Met Glu Ile Ty #r Gly Val Cys Lys Glu    130               #   135               #   140 Cys Gln Asp Lys 145<210> SEQ ID NO 7 <211> LENGTH: 600 <212> TYPE: DNA<213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 7tgagaaaagc ttgcatttta ttgagaaaac tgttagtttt aattgtaaag tt#tgaaataa     60tttgtaatga ttttaattat tagtagggga gtggacatcg ttggaagaac ga#ttaaatcg    120cgttaagcaa caattacaac aatcatcata taagctaacg ccacaacgcg aa#gctactgt    180tagagttcta attgaaaatg aaaaagatca tctaagtgct gaagacgtat at#ctgaaagt    240aaaagataaa gcgcctgaaa ttggcttggc gacagtatac agaacgttag ag#ttgttagc    300tgaactaaaa gttgtcgaca aaattaactt tggtgatggc gtcgctcgtt tt#gatttaag    360aaaagaaggc gcaaaacatt tccaccatca tttagtatgt atggaatgtg gt#cgtgtaga    420tgaaatcgat gaagatttgt taccagaagt tgaaaatcga gttgaaaatg ag#ttcaattt    480taaaatttta gatcatcgtt taactttcca tggtgtgtgt gaaacgtgcc aa#gctaaagg    540taaaggatag taaattgcgt aggttaaatt aaccttcgct ttttttagag gt#gtggttat    600 <210> SEQ ID NO 8 <211> LENGTH: 149 <212> TYPE: PRT<213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 8Leu Glu Glu Arg Leu Asn Arg Val Lys Gln Gl #n Leu Gln Gln Ser Ser  1               5  #                 10  #                 15Tyr Lys Leu Thr Pro Gln Arg Glu Ala Thr Va #l Arg Val Leu Ile Glu             20      #             25      #             30Asn Glu Lys Asp His Leu Ser Ala Glu Asp Va #l Tyr Leu Lys Val Lys         35          #         40          #         45Asp Lys Ala Pro Glu Ile Gly Leu Ala Thr Va #l Tyr Arg Thr Leu Glu     50              #     55              #     60Leu Leu Ala Glu Leu Lys Val Val Asp Lys Il #e Asn Phe Gly Asp Gly 65                  # 70                  # 75                  # 80Val Ala Arg Phe Asp Leu Arg Lys Glu Gly Al #a Lys His Phe His His                 85  #                 90  #                 95His Leu Val Cys Met Glu Cys Gly Arg Val As #p Glu Ile Asp Glu Asp            100       #           105       #           110Leu Leu Pro Glu Val Glu Asn Arg Val Glu As #n Glu Phe Asn Phe Lys        115           #       120           #       125Ile Leu Asp His Arg Leu Thr Phe His Gly Va #l Cys Glu Thr Cys Gln    130               #   135               #   140 Ala Lys Gly Lys Gly145 <210> SEQ ID NO 9 <211> LENGTH: 1647 <212> TYPE: DNA<213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 9gtaaatatac ctctttaatt aatttattca atagaactgg tataataaaa ta#aatctcat     60taggcactta agtaaattta acatataaaa aggaacgttt atgactacta aa#aaactgta    120ttttctatcc atttctatta tcattttagt cgccatttca attgctatat at#ataacatt    180aaatagcaat acgaagacac ggttaaccaa tgattcgcaa caacaaatag at#acaattat    240cgagcatgat ttacaaaagg gacacattcc tggagcatca attttaatag ta#aaaaatgg    300caaagttttt ttaaataaag gttatggtta tcaagatgtt gataaaaaag tc#aaagcttc    360tcccacaaca aagtatgaaa ttgcttctaa tacgaaagct ttcacaggtc tt#gcaatttt    420aaaattagct caagaaggtc gattaaactt aaatgatgcc gtatccaaac at#gtgcctca    480ttttaaaatg aactataatg gtcaaaatga aactattacg attaagcaac tt#ttggctca    540aacaagtggt atacctagtg atattacaag cgaagattct gtgacaagca aa#aataatcg    600tttaaatgat gtaacccatg caattatggg tgatgaatta catcataagc cc#ggagaaga    660atttgaatac tcaaatatga actatgattt attaggttta attatccaaa ac#gttacgaa    720gcaatcctat acaaaatata ttacaaattc atggctcaag cctttgcata tg#acacatac    780atcattcaaa caaaccaatt acaaatcaaa acatgatgct attggctatg aa#ttacaagg    840ttcgacacct gtcgtctcta aacctgaatt taacctttgg gatacaccat ca#gcatatat    900gatgacatca actgaagatt tggaacattg gataaaattc caacttaatc ca#cctgataa    960atacaaatca ttagttcaac aatcacataa aaatttatct tcaacaattg gt#gaacctaa   1020tgccaatgca tatgcttccg gctggtttac caataatgat gaacatttag tg#tttcattc   1080aggaacgcta gataactttt catcatttat tttactaaat ccaaaacaaa at#tatggaat   1140tgttgtactt gcaaatctaa attcggaata tgtacccaaa ttagttgagc at#cttaatac   1200acaaattgta aatcacaagc gatattcgac ggttgcgtct atgctcaatc aa#tataaaga   1260tcaatttaat attgttaccg ttttgatgac aacacttatt ttattagcat tt#atattctc   1320agcttatcgt gcttggcaaa tgcgccatgg tcaaattctt ttgcgtagat ca#aaacggat   1380tgctgtattg agttggttat cattatgtat atgtatcgct ttagcgctca ta#ttatatgc   1440attaccatat ctcattctcg gtagcaataa ttggtctttt gtactgactt gg#ctaccaat   1500agaaattaaa ttagcactaa tcacaacatt aattgcatta ttcagtacat ta#attgtaat   1560tctgttattc cttcatacaa agataacgaa gacataataa aaaagacttg tt#cgagccgt   1620 gcgtttgata atatatcatc cacgatt          #                   #           1647 <210> SEQ ID NO 10<211> LENGTH: 498 <212> TYPE: PRT <213> ORGANISM: Staphylococcus aureus<400> SEQUENCE: 10 Met Thr Thr Lys Lys Leu Tyr Phe Leu Ser Il#e Ser Ile Ile Ile Leu   1               5  #                 10 #                 15 Val Ala Ile Ser Ile Ala Ile Tyr Ile Thr Le#u Asn Ser Asn Thr Lys              20      #             25     #             30 Thr Arg Leu Thr Asn Asp Ser Gln Gln Gln Il#e Asp Thr Ile Ile Glu          35          #         40         #         45 His Asp Leu Gln Lys Gly His Ile Pro Gly Al#a Ser Ile Leu Ile Val      50              #     55             #     60 Lys Asn Gly Lys Val Phe Leu Asn Lys Gly Ty#r Gly Tyr Gln Asp Val  65                  # 70                 # 75                  # 80 Asp Lys Lys Val Lys Ala Ser Pro Thr Thr Ly#s Tyr Glu Ile Ala Ser                  85  #                 90 #                 95 Asn Thr Lys Ala Phe Thr Gly Leu Ala Ile Le#u Lys Leu Ala Gln Glu             100       #           105      #           110 Gly Arg Leu Asn Leu Asn Asp Ala Val Ser Ly#s His Val Pro His Phe         115           #       120          #       125 Lys Met Asn Tyr Asn Gly Gln Asn Glu Thr Il#e Thr Ile Lys Gln Leu     130               #   135              #   140 Leu Ala Gln Thr Ser Gly Ile Pro Ser Asp Il#e Thr Ser Glu Asp Ser 145                 1 #50                 1#55                 1 #60 Val Thr Ser Lys Asn Asn Arg Leu Asn Asp Va#l Thr His Ala Ile Met                 165   #               170  #               175 Gly Asp Glu Leu His His Lys Pro Gly Glu Gl#u Phe Glu Tyr Ser Asn             180       #           185      #           190 Met Asn Tyr Asp Leu Leu Gly Leu Ile Ile Gl#n Asn Val Thr Lys Gln         195           #       200          #       205 Ser Tyr Thr Lys Tyr Ile Thr Asn Ser Trp Le#u Lys Pro Leu His Met     210               #   215              #   220 Thr His Thr Ser Phe Lys Gln Thr Asn Tyr Ly#s Ser Lys His Asp Ala 225                 2 #30                 2#35                 2 #40 Ile Gly Tyr Glu Leu Gln Gly Ser Thr Pro Va#l Val Ser Lys Pro Glu                 245   #               250  #               255 Phe Asn Leu Trp Asp Thr Pro Ser Ala Tyr Me#t Met Thr Ser Thr Glu             260       #           265      #           270 Asp Leu Glu His Trp Ile Lys Phe Gln Leu As#n Pro Pro Asp Lys Tyr         275           #       280          #       285 Lys Ser Leu Val Gln Gln Ser His Lys Asn Le#u Ser Ser Thr Ile Gly     290               #   295              #   300 Glu Pro Asn Ala Asn Ala Tyr Ala Ser Gly Tr#p Phe Thr Asn Asn Asp 305                 3 #10                 3#15                 3 #20 Glu His Leu Val Phe His Ser Gly Thr Leu As#p Asn Phe Ser Ser Phe                 325   #               330  #               335 Ile Leu Leu Asn Pro Lys Gln Asn Tyr Gly Il#e Val Val Leu Ala Asn             340       #           345      #           350 Leu Asn Ser Glu Tyr Val Pro Lys Leu Val Gl#u His Leu Asn Thr Gln         355           #       360          #       365 Ile Val Asn His Lys Arg Tyr Ser Thr Val Al#a Ser Met Leu Asn Gln     370               #   375              #   380 Tyr Lys Asp Gln Phe Asn Ile Val Thr Val Le#u Met Thr Thr Leu Ile 385                 3 #90                 3#95                 4 #00 Leu Leu Ala Phe Ile Phe Ser Ala Tyr Arg Al#a Trp Gln Met Arg His                 405   #               410  #               415 Gly Gln Ile Leu Leu Arg Arg Ser Lys Arg Il#e Ala Val Leu Ser Trp             420       #           425      #           430 Leu Ser Leu Cys Ile Cys Ile Ala Leu Ala Le#u Ile Leu Tyr Ala Leu         435           #       440          #       445 Pro Tyr Leu Ile Leu Gly Ser Asn Asn Trp Se#r Phe Val Leu Thr Trp     450               #   455              #   460 Leu Pro Ile Glu Ile Lys Leu Ala Leu Ile Th#r Thr Leu Ile Ala Leu 465                 4 #70                 4#75                 4 #80 Phe Ser Thr Leu Ile Val Ile Leu Leu Phe Le#u His Thr Lys Ile Thr                 485   #               490  #               495 Lys Thr <210> SEQ ID NO 11 <211> LENGTH: 2226<212> TYPE: DNA <213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 11ctcttaaatg agaccgttat ttttttgtca aaaagataga aataatttct aa#attcatat     60atgatttaaa gtgaaagact ttgaatagag gtaggtagtt ttgttaaaaa ga#ctaaaaga    120aaaatcaaat gatgaaatcg ttcaaaatac cattaacaag agaattaact tt#atatttgg    180tgtgattgta tttatttttg cagtactagt actacgttta ggttatttac aa#atcgcaca    240aggctcacat tataaacaaa ttataaaaaa tgatgaaaac attacagtga at#gagtctgt    300gccaagaggt cgtattttag acagaaatgg gaaagtttta gttgataatg ct#tctaaaat    360ggctattaca tatactaggg gtcgaaaaac aacacaatcg gaaatgttgg at#acggctga    420aaagttatca aagctaatca agatggatac taagaaaatt acagaacgtg at#aagaaaga    480tttctggatt cagttgcatc ctaaaaaagc aaaagcaatg atgacaaaag aa#caagctat    540gttagcagat ggaagtatta aacaagatca atatgataaa caactgttat cg#aaaatcgg    600aaaatcacaa ttagatgaat tgtcttctaa agatttacaa gttttagcta tt#tttcgaga    660gatgaatgca ggaacagttt tagatccaca aatgataaaa aatgaagatg tc#agtgaaaa    720agagtatgca gcagtttctc agcaactttc caaattacca ggtgttaaca cg#tctatgga    780ttgggataga aaatatccat atggcgatac tttaagaggt atattcggag at#gtatcgac    840acctgctgaa ggtattccaa aagaattgac agaacattac ttatccaaag ga#tattcacg    900caatgatcgt gttggaaaat cttacctaga atatcaatat gaagatgtat tg#cgtggtaa    960gaagaaagaa atgaaataca caacggacaa atctggtaaa gttacatctt ca#gaagtgtt   1020aaatcctggc gctcgcggtc aagatttgaa attaacgatc gatatagatc tt#caaaaaga   1080agtagaagca ttattagata aacaaattaa gaagcttcgc agtcaaggtg cc#aaagatat   1140ggataatgca atgatggttg tacaaaatcc taaaaatgga gacattcttg cg#cttgccgg   1200aaagcagatt aataagagtg gtaaaatgac tgattatgac attggtacgt tt#acttctca   1260atttgcggtt ggatcttctg taaaaggtgg aacattatta gccggttatc ag#aataaagc   1320tatcaaagtt ggagaaacaa tggtcgatga accattacat ttccaaggtg gt#ttgacaaa   1380acgatcatac ttcaataaaa acgggcatgt aactattaat gataagcaag ct#ttgatgca   1440ttcatcaaac gtatatatgt ttaaaacagc attaaaatta gcgggagacc ct#tattattc   1500tggtatggct ttaccttcag acataagttc acctgcccaa aagctaagaa ga#ggattaaa   1560tcaagtaggc ttaggtgtga aaacagggat agatttacca aatgaaacaa ga#ggtcaaat   1620cgaaccatta acaaataatc caggtaatta tctagattta tcaattggtc aa#tatgatac   1680ctatacacca ttacaattat cacaatatgt ttcaactata gcgaatgatg gt#tatagaat   1740acagccacac attggattaa cgattcatga atcaactaat aaagatgagg tt#ggtccact   1800caagaagaaa attaatggca ctgtcttgaa caaggttaat aatactgaaa ag#gaaatcaa   1860acaaattcaa gaaggattca aaatggcatt taatgataaa gatggtactg ga#tatgttag   1920ttttaaagat acagtagtac ctactgctgg taaaacgggt accgcagaag tg#ttccaaaa   1980cggagagcca agagttaact ctacttatat aggatacgcg ccaattgatg at#ccaaaatt   2040agcgttttca attgtatata caaatcagcc tgtaccacca ccatggttaa ca#ggtggaga   2100cttaggtaga gatgtaatta actactactt taagcagtta ggtaaagatg at#aaaaataa   2160agacaaagac aaataaaatt taacctgacg attgtgtagc gcatggttgt aa#aattttaa   2220 ctttgc                  #                  #                   #         2226 <210> SEQ ID NO 12 <211> LENGTH: 691<212> TYPE: PRT <213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 12Leu Leu Lys Arg Leu Lys Glu Lys Ser Asn As #p Glu Ile Val Gln Asn  1               5  #                 10  #                 15Thr Ile Asn Lys Arg Ile Asn Phe Ile Phe Gl #y Val Ile Val Phe Ile             20      #             25      #             30Phe Ala Val Leu Val Leu Arg Leu Gly Tyr Le #u Gln Ile Ala Gln Gly         35          #         40          #         45Ser His Tyr Lys Gln Ile Ile Lys Asn Asp Gl #u Asn Ile Thr Val Asn     50              #     55              #     60Glu Ser Val Pro Arg Gly Arg Ile Leu Asp Ar #g Asn Gly Lys Val Leu 65                  # 70                  # 75                  # 80Val Asp Asn Ala Ser Lys Met Ala Ile Thr Ty #r Thr Arg Gly Arg Lys                 85  #                 90  #                 95Thr Thr Gln Ser Glu Met Leu Asp Thr Ala Gl #u Lys Leu Ser Lys Leu            100       #           105       #           110Ile Lys Met Asp Thr Lys Lys Ile Thr Glu Ar #g Asp Lys Lys Asp Phe        115           #       120           #       125Trp Ile Gln Leu His Pro Lys Lys Ala Lys Al #a Met Met Thr Lys Glu    130               #   135               #   140Gln Ala Met Leu Ala Asp Gly Ser Ile Lys Gl #n Asp Gln Tyr Asp Lys145                 1 #50                 1 #55                 1 #60Gln Leu Leu Ser Lys Ile Gly Lys Ser Gln Le #u Asp Glu Leu Ser Ser                165   #               170   #               175Lys Asp Leu Gln Val Leu Ala Ile Phe Arg Gl #u Met Asn Ala Gly Thr            180       #           185       #           190Val Leu Asp Pro Gln Met Ile Lys Asn Glu As #p Val Ser Glu Lys Glu        195           #       200           #       205Tyr Ala Ala Val Ser Gln Gln Leu Ser Lys Le #u Pro Gly Val Asn Thr    210               #   215               #   220Ser Met Asp Trp Asp Arg Lys Tyr Pro Tyr Gl #y Asp Thr Leu Arg Gly225                 2 #30                 2 #35                 2 #40Ile Phe Gly Asp Val Ser Thr Pro Ala Glu Gl #y Ile Pro Lys Glu Leu                245   #               250   #               255Thr Glu His Tyr Leu Ser Lys Gly Tyr Ser Ar #g Asn Asp Arg Val Gly            260       #           265       #           270Lys Ser Tyr Leu Glu Tyr Gln Tyr Glu Asp Va #l Leu Arg Gly Lys Lys        275           #       280           #       285Lys Glu Met Lys Tyr Thr Thr Asp Lys Ser Gl #y Lys Val Thr Ser Ser    290               #   295               #   300Glu Val Leu Asn Pro Gly Ala Arg Gly Gln As #p Leu Lys Leu Thr Ile305                 3 #10                 3 #15                 3 #20Asp Ile Asp Leu Gln Lys Glu Val Glu Ala Le #u Leu Asp Lys Gln Ile                325   #               330   #               335Lys Lys Leu Arg Ser Gln Gly Ala Lys Asp Me #t Asp Asn Ala Met Met            340       #           345       #           350Val Val Gln Asn Pro Lys Asn Gly Asp Ile Le #u Ala Leu Ala Gly Lys        355           #       360           #       365Gln Ile Asn Lys Ser Gly Lys Met Thr Asp Ty #r Asp Ile Gly Thr Phe    370               #   375               #   380Thr Ser Gln Phe Ala Val Gly Ser Ser Val Ly #s Gly Gly Thr Leu Leu385                 3 #90                 3 #95                 4 #00Ala Gly Tyr Gln Asn Lys Ala Ile Lys Val Gl #y Glu Thr Met Val Asp                405   #               410   #               415Glu Pro Leu His Phe Gln Gly Gly Leu Thr Ly #s Arg Ser Tyr Phe Asn            420       #           425       #           430Lys Asn Gly His Val Thr Ile Asn Asp Lys Gl #n Ala Leu Met His Ser        435           #       440           #       445Ser Asn Val Tyr Met Phe Lys Thr Ala Leu Ly #s Leu Ala Gly Asp Pro    450               #   455               #   460Tyr Tyr Ser Gly Met Ala Leu Pro Ser Asp Il #e Ser Ser Pro Ala Gln465                 4 #70                 4 #75                 4 #80Lys Leu Arg Arg Gly Leu Asn Gln Val Gly Le #u Gly Val Lys Thr Gly                485   #               490   #               495Ile Asp Leu Pro Asn Glu Thr Arg Gly Gln Il #e Glu Pro Leu Thr Asn            500       #           505       #           510Asn Pro Gly Asn Tyr Leu Asp Leu Ser Ile Gl #y Gln Tyr Asp Thr Tyr        515           #       520           #       525Thr Pro Leu Gln Leu Ser Gln Tyr Val Ser Th #r Ile Ala Asn Asp Gly    530               #   535               #   540Tyr Arg Ile Gln Pro His Ile Gly Leu Thr Il #e His Glu Ser Thr Asn545                 5 #50                 5 #55                 5 #60Lys Asp Glu Val Gly Pro Leu Lys Lys Lys Il #e Asn Gly Thr Val Leu                565   #               570   #               575Asn Lys Val Asn Asn Thr Glu Lys Glu Ile Ly #s Gln Ile Gln Glu Gly            580       #           585       #           590Phe Lys Met Ala Phe Asn Asp Lys Asp Gly Th #r Gly Tyr Val Ser Phe        595           #       600           #       605Lys Asp Thr Val Val Pro Thr Ala Gly Lys Th #r Gly Thr Ala Glu Val    610               #   615               #   620Phe Gln Asn Gly Glu Pro Arg Val Asn Ser Th #r Tyr Ile Gly Tyr Ala625                 6 #30                 6 #35                 6 #40Pro Ile Asp Asp Pro Lys Leu Ala Phe Ser Il #e Val Tyr Thr Asn Gln                645   #               650   #               655Pro Val Pro Pro Pro Trp Leu Thr Gly Gly As #p Leu Gly Arg Asp Val            660       #           665       #           670Ile Asn Tyr Tyr Phe Lys Gln Leu Gly Lys As #p Asp Lys Asn Lys Asp        675           #       680           #       685 Lys Asp Lys    690 <210> SEQ ID NO 13 <211> LENGTH: 1056 <212> TYPE: DNA<213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 13tcctattcct tatgcatttc ccctaattat aattaacgtt aaaataaaag tc#aaattgcc     60ttaaatatgg tatactataa cgtaatttag gaggttaaag atgacgaatc aa#gacaacaa    120tcatcaattg aatcatcgta tatatcattt tgaaaagata tataaagcta tc#aaacatgt    180cattgtttac atatttatga ttttcattgc catcgttgct atcgctgtga tt#gcgatgtc    240tttatatttt catcatttaa ctaaaacgtc cgactcatta tcagatgatg ct#ttaataaa    300aaaagttcga caaatacctg gcgatgaatt attagatcat aataacaaaa at#ttattata    360tgagtataac cattctcaaa actcactcat tataggccct aaaacatcaa gt#ccaaatgt    420cattaaagca ttaacgtcat ctgaagacac tttattttat aaacatgatg gc#atcttacc    480aaaggcgatt ttaagagcaa tgatacaaga tatttttaat actgatcaaa gt#tcaggtgg    540tagcacaatt acacaacaac ttgttaaaaa tcaagttctt accaacgaaa aa#acatatag    600tagaaaagca aatgaacttc gcctagcaat tagattagaa cacctactct ca#aaagatga    660aattatatat acatatttaa atatagttcc cttcggtaga gattataatg gc#gctaatat    720ttccggaatt gcatccgctt catatagtct atttggtatt ccaccaaaag at#ttatcaat    780tgcacaatct gcatacctta tcggtttgtt gcaaagccca tatggctata ca#ccctacga    840aaaagatgga acgttaaaat cggataaaga tttgaaatat agtattcaaa ga#caacatta    900tgtattaaag cgtatgttaa tcgaagatca aatcactgaa aaagaataca ac#gacgcatt    960aaaatatgat attaaatcac atttgttaaa tcgaaaaaag cgttaattga tg#ctcacttt   1020 ttaaagtaac cacaacaatg aatccaaata ttaaaa      #                   #     1056 <210> SEQ ID NO 14 <211> LENGTH: 301<212> TYPE: PRT <213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 14Met Thr Asn Gln Asp Asn Asn His Gln Leu As #n His Arg Ile Tyr His  1               5  #                 10  #                 15Phe Glu Lys Ile Tyr Lys Ala Ile Lys His Va #l Ile Val Tyr Ile Phe             20      #             25      #             30Met Ile Phe Ile Ala Ile Val Ala Ile Ala Va #l Ile Ala Met Ser Leu         35          #         40          #         45Tyr Phe His His Leu Thr Lys Thr Ser Asp Se #r Leu Ser Asp Asp Ala     50              #     55              #     60Leu Ile Lys Lys Val Arg Gln Ile Pro Gly As #p Glu Leu Leu Asp His 65                  # 70                  # 75                  # 80Asn Asn Lys Asn Leu Leu Tyr Glu Tyr Asn Hi #s Ser Gln Asn Ser Leu                 85  #                 90  #                 95Ile Ile Gly Pro Lys Thr Ser Ser Pro Asn Va #l Ile Lys Ala Leu Thr            100       #           105       #           110Ser Ser Glu Asp Thr Leu Phe Tyr Lys His As #p Gly Ile Leu Pro Lys        115           #       120           #       125Ala Ile Leu Arg Ala Met Ile Gln Asp Ile Ph #e Asn Thr Asp Gln Ser    130               #   135               #   140Ser Gly Gly Ser Thr Ile Thr Gln Gln Leu Va #l Lys Asn Gln Val Leu145                 1 #50                 1 #55                 1 #60Thr Asn Glu Lys Thr Tyr Ser Arg Lys Ala As #n Glu Leu Arg Leu Ala                165   #               170   #               175Ile Arg Leu Glu His Leu Leu Ser Lys Asp Gl #u Ile Ile Tyr Thr Tyr            180       #           185       #           190Leu Asn Ile Val Pro Phe Gly Arg Asp Tyr As #n Gly Ala Asn Ile Ser        195           #       200           #       205Gly Ile Ala Ser Ala Ser Tyr Ser Leu Phe Gl #y Ile Pro Pro Lys Asp    210               #   215               #   220Leu Ser Ile Ala Gln Ser Ala Tyr Leu Ile Gl #y Leu Leu Gln Ser Pro225                 2 #30                 2 #35                 2 #40Tyr Gly Tyr Thr Pro Tyr Glu Lys Asp Gly Th #r Leu Lys Ser Asp Lys                245   #               250   #               255Asp Leu Lys Tyr Ser Ile Gln Arg Gln His Ty #r Val Leu Lys Arg Met            260       #           265       #           270Leu Ile Glu Asp Gln Ile Thr Glu Lys Glu Ty #r Asn Asp Ala Leu Lys        275           #       280           #       285Tyr Asp Ile Lys Ser His Leu Leu Asn Arg Ly #s Lys Arg    290               #   295               #   300 <210> SEQ ID NO 15<211> LENGTH: 999 <212> TYPE: DNA <213> ORGANISM: Staphylococcus aureus<400> SEQUENCE: 15tagtcaatga ataaagtaat taaaatgctt gttgttacgc ttgctttcct ac#ttgtttta     60gcaggatgta gtgggaattc aaataaacaa tcatctgata acaaagataa gg#aaacaact    120tcaattaaac atgcaatggg tacaactgaa attaaaggga aaccaaagcg tg#ttgttacg    180ctatatcaag gtgccactga cgtcgctgta tctttaggtg ttaaacctgt ag#gtgctgta    240gaatcatgga cacaaaaacc gaaattcgaa tacataaaaa atgatttaaa ag#atactaag    300attgtaggtc aagaacctgc acctaactta gaggaaatct ctaaattaaa ac#cggactta    360attgtcgcgt caaaagttag aaatgaaaaa gtttacgatc aattatctaa aa#tcgcacca    420acagtttcta ctgatacagt tttcaaattc aaagatacaa ctaagttaat gg#ggaaagct    480ttagggaaag aaaaagaagc tgaagattta cttaaaaagt acgatgataa ag#tagctgca    540ttccaaaaag atgcaaaagc aaagtataaa gatgcatggc cattgaaagc tt#cagttgtt    600aacttccgtg ctgatcatac aagaatttat gctggtggat atgctggtga aa#tcttaaat    660gatttaggat tcaaacgtaa taaagactta caaaaacaag ttgataatgg ta#aagatatt    720atccaactta catctaaaga aagcattcca ttaatgaacg ctgatcatat tt#ttgtagta    780aaatcagatc caaatgcgaa agatgctgca ttagttaaaa agactgaaag cg#aatggact    840tcaagtaaag agtggaaaaa tttagacgca gttaaaaaca accaagtatc tg#atgattta    900gatgaaatca cttggaactt agctggcgga tataaatctt cattaaaact ta#ttgacgat    960 ttatatgaaa agttaaatat tgaaaaacaa tcaaaataa      #                   #   999 <210> SEQ ID NO 16 <211> LENGTH: 330<212> TYPE: PRT <213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 16Met Asn Lys Val Ile Lys Met Leu Val Val Th #r Leu Ala Phe Leu Leu  1               5  #                 10  #                 15Val Leu Ala Gly Cys Ser Gly Asn Ser Asn Ly #s Gln Ser Ser Asp Asn             20      #             25      #             30Lys Asp Lys Glu Thr Thr Ser Ile Lys His Al #a Met Gly Thr Thr Glu         35          #         40          #         45Ile Lys Gly Lys Pro Lys Arg Val Val Thr Le #u Tyr Gln Gly Ala Thr     50              #     55              #     60Asp Val Ala Val Ser Leu Gly Val Lys Pro Va #l Gly Ala Val Glu Ser 65                  # 70                  # 75                  # 80Trp Thr Gln Lys Pro Lys Phe Glu Tyr Ile Ly #s Asn Asp Leu Lys Asp                 85  #                 90  #                 95Thr Lys Ile Val Gly Gln Glu Pro Ala Pro As #n Leu Glu Glu Ile Ser            100       #           105       #           110Lys Leu Lys Pro Asp Leu Ile Val Ala Ser Ly #s Val Arg Asn Glu Lys        115           #       120           #       125Val Tyr Asp Gln Leu Ser Lys Ile Ala Pro Th #r Val Ser Thr Asp Thr    130               #   135               #   140Val Phe Lys Phe Lys Asp Thr Thr Lys Leu Me #t Gly Lys Ala Leu Gly145                 1 #50                 1 #55                 1 #60Lys Glu Lys Glu Ala Glu Asp Leu Leu Lys Ly #s Tyr Asp Asp Lys Val                165   #               170   #               175Ala Ala Phe Gln Lys Asp Ala Lys Ala Lys Ty #r Lys Asp Ala Trp Pro            180       #           185       #           190Leu Lys Ala Ser Val Val Asn Phe Arg Ala As #p His Thr Arg Ile Tyr        195           #       200           #       205Ala Gly Gly Tyr Ala Gly Glu Ile Leu Asn As #p Leu Gly Phe Lys Arg    210               #   215               #   220Asn Lys Asp Leu Gln Lys Gln Val Asp Asn Gl #y Lys Asp Ile Ile Gln225                 2 #30                 2 #35                 2 #40Leu Thr Ser Lys Glu Ser Ile Pro Leu Met As #n Ala Asp His Ile Phe                245   #               250   #               255Val Val Lys Ser Asp Pro Asn Ala Lys Asp Al #a Ala Leu Val Lys Lys            260       #           265       #           270Thr Glu Ser Glu Trp Thr Ser Ser Lys Glu Tr #p Lys Asn Leu Asp Ala        275           #       280           #       285Val Lys Asn Asn Gln Val Ser Asp Asp Leu As #p Glu Ile Thr Trp Asn    290               #   295               #   300Leu Ala Gly Gly Tyr Lys Ser Ser Leu Lys Le #u Ile Asp Asp Leu Tyr305                 3 #10                 3 #15                 3 #20Glu Lys Leu Asn Ile Glu Lys Gln Ser Lys                 325  #               330 <210> SEQ ID NO 17 <211> LENGTH: 1014<212> TYPE: DNA <213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 17taattaagga gttttacgat gctacttaaa ccaaaatacc aaatcgttat tg#ctggttta     60tgtcttgcaa tagtagctat cttaagttta atgattggaa atacgcttgt gt#caccaggt    120acggtgatac aggcgttatt caactttgat agtgaaaacg atttacatga tg#ttgtcact    180ggtgcacggg cgtcgagaac aatcattgcg ttattgactg gtgctgccct tg#ctgtctca    240ggtttgttga tgcaagcact tacacgaaac ccaatagcct caccagggct tt#tcggtgtc    300aatgcaggcg cagtattttt tgtcattttt agtattacat ttatccaaat tc#aatctttt    360aaaatgattg tagttattgc atttttgggg gctattgttg ttactgtatt ag#ttgttgca    420ctaggtatgt ttagacaaac actattctca cctcaccgtg tcattttggc ag#gtgctgcg    480attgcgatgc tatttacagc ctttactcaa ggcatactta ttatgaacga aa#cagactta    540caaggcctat tattttggtt aagtggctcc gtttcattac gtaatatttg gg#atatccca    600tggattattc cgcttgtatt gatacttatt ttaattgcat ttagcatggc tg#cacacatc    660aacatcttga tgacaagtga cgacattgca accggcctcg gtcaaaacat aa#aattaatc    720aaatggatga ttattatgct catcagtatg ttagccggta tttcggtagc cg#tagctgga    780tcaatcgtct ttgtgggtct tatcgtaccg aatattagca aacgattatt ac#caccaaac    840tataagtatt taattccttt tactgcatta gctggagcaa tcctaatgat ca#tttcagac    900attgttgctc gtataataat taagccacta gagttgccta tcggtgtcgt ta#ccgctgtc    960attggcgcta ttgtcttaat ctatattatg aagaaaggac gtcaacgctt at#ga         1014 <210> SEQ ID NO 18 <211> LENGTH: 331 <212> TYPE: PRT<213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 18Met Leu Leu Lys Pro Lys Tyr Gln Ile Val Il #e Ala Gly Leu Cys Leu  1               5  #                 10  #                 15Ala Ile Val Ala Ile Leu Ser Leu Met Ile Gl #y Asn Thr Leu Val Ser             20      #             25      #             30Pro Gly Thr Val Ile Gln Ala Leu Phe Asn Ph #e Asp Ser Glu Asn Asp         35          #         40          #         45Leu His Asp Val Val Thr Gly Ala Arg Ala Se #r Arg Thr Ile Ile Ala     50              #     55              #     60Leu Leu Thr Gly Ala Ala Leu Ala Val Ser Gl #y Leu Leu Met Gln Ala 65                  # 70                  # 75                  # 80Leu Thr Arg Asn Pro Ile Ala Ser Pro Gly Le #u Phe Gly Val Asn Ala                 85  #                 90  #                 95Gly Ala Val Phe Phe Val Ile Phe Ser Ile Th #r Phe Ile Gln Ile Gln            100       #           105       #           110Ser Phe Lys Met Ile Val Val Ile Ala Phe Le #u Gly Ala Ile Val Val        115           #       120           #       125Thr Val Leu Val Val Ala Leu Gly Met Phe Ar #g Gln Thr Leu Phe Ser    130               #   135               #   140Pro His Arg Val Ile Leu Ala Gly Ala Ala Il #e Ala Met Leu Phe Thr145                 1 #50                 1 #55                 1 #60Ala Phe Thr Gln Gly Ile Leu Ile Met Asn Gl #u Thr Asp Leu Gln Gly                165   #               170   #               175Leu Leu Phe Trp Leu Ser Gly Ser Val Ser Le #u Arg Asn Ile Trp Asp            180       #           185       #           190Ile Pro Trp Ile Ile Pro Leu Val Leu Ile Le #u Ile Leu Ile Ala Phe        195           #       200           #       205Ser Met Ala Ala His Ile Asn Ile Leu Met Th #r Ser Asp Asp Ile Ala    210               #   215               #   220Thr Gly Leu Gly Gln Asn Ile Lys Leu Ile Ly #s Trp Met Ile Ile Met225                 2 #30                 2 #35                 2 #40Leu Ile Ser Met Leu Ala Gly Ile Ser Val Al #a Val Ala Gly Ser Ile                245   #               250   #               255Val Phe Val Gly Leu Ile Val Pro Asn Ile Se #r Lys Arg Leu Leu Pro            260       #           265       #           270Pro Asn Tyr Lys Tyr Leu Ile Pro Phe Thr Al #a Leu Ala Gly Ala Ile        275           #       280           #       285Leu Met Ile Ile Ser Asp Ile Val Ala Arg Il #e Ile Ile Lys Pro Leu    290               #   295               #   300Glu Leu Pro Ile Gly Val Val Thr Ala Val Il #e Gly Ala Ile Val Leu305                 3 #10                 3 #15                 3 #20Ile Tyr Ile Met Lys Lys Gly Arg Gln Arg Le #u                 325  #               330 <210> SEQ ID NO 19 <211> LENGTH: 1089<212> TYPE: DNA <213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 19taagccacta gagttgccta tcggtgtcgt taccgctgtc attggcgcta tt#gtcttaat     60ctatattatg aagaaaggac gtcaacgctt atgaccgaaa agattaataa aa#aagacaat    120taccatctca tcttcgcgtt aatcttttta gccatcgttt cagtggtaag ta#tgatgatt    180ggttcaagct ttataccatt acaacgcgta ctgatgtact ttataaatcc aa#atgacagt    240atggatcaat tcactttaga agtattacgc ttacctcgca ttacacttgc ga#ttttagca    300ggtgccgcac taggaatgag tggtttaatg ttgcaaaatg tattaaaaaa tc#caattgcc    360tcacctgata ttatcggtat cacaggtggt gctagcttaa gtgctgttgt ct#ttattgca    420tttttcagcc atttaacaat acatttactt ccactatttg cagtattagg tg#gcgcagtt    480gcaatgatga tactattagt gtttcaaacg aaaggacaaa tacgcccgac aa#cactcata    540atcatcggta tttcgatgca aacgttgttt attgcgcttg tccaaggatt ac#tcattaca    600acgaagcaat tatctgctgc caaagcttat acatggctag tcggaagtct tt#acggtgct    660acgtttaaag atacaatcat tttgggtatg gttattttag ctgttgtgcc gt#tgttattt    720cttgttatac caaaaatgaa aatatctata cttgatgacc ctgtagcgat tg#gcttaggc    780ttacatgtac aacgtatgaa actaatccaa ttaatcactt ctactatact cg#tatctatg    840gcaatcagtt tagtaggtaa cattgggttt gtcggtttaa tcgcaccaca ta#tcgcgaaa    900acaatcgttc gcggaagtta tgctaaaaag ttactaatgt cagcaatgat tg#gtgccata    960tcaattgtta ttgcagactt aattgggcgt accttattct tgcctaaaga ag#tgccagca   1020ggtgtattta ttgctgcttt tggtgcccca ttcttcatat acttattatt aa#ccgtgaaa   1080 aagttataa                 #                  #                   #       1089 <210> SEQ ID NO 20 <211> LENGTH: 332<212> TYPE: PRT <213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 20Met Thr Glu Lys Ile Asn Lys Lys Asp Asn Ty #r His Leu Ile Phe Ala  1               5  #                 10  #                 15Leu Ile Phe Leu Ala Ile Val Ser Val Val Se #r Met Met Ile Gly Ser             20      #             25      #             30Ser Phe Ile Pro Leu Gln Arg Val Leu Met Ty #r Phe Ile Asn Pro Asn         35          #         40          #         45Asp Ser Met Asp Gln Phe Thr Leu Glu Val Le #u Arg Leu Pro Arg Ile     50              #     55              #     60Thr Leu Ala Ile Leu Ala Gly Ala Ala Leu Gl #y Met Ser Gly Leu Met 65                  # 70                  # 75                  # 80Leu Gln Asn Val Leu Lys Asn Pro Ile Ala Se #r Pro Asp Ile Ile Gly                 85  #                 90  #                 95Ile Thr Gly Gly Ala Ser Leu Ser Ala Val Va #l Phe Ile Ala Phe Phe            100       #           105       #           110Ser His Leu Thr Ile His Leu Leu Pro Leu Ph #e Ala Val Leu Gly Gly        115           #       120           #       125Ala Val Ala Met Met Ile Leu Leu Val Phe Gl #n Thr Lys Gly Gln Ile    130               #   135               #   140Arg Pro Thr Thr Leu Ile Ile Ile Gly Ile Se #r Met Gln Thr Leu Phe145                 1 #50                 1 #55                 1 #60Ile Ala Leu Val Gln Gly Leu Leu Ile Thr Th #r Lys Gln Leu Ser Ala                165   #               170   #               175Ala Lys Ala Tyr Thr Trp Leu Val Gly Ser Le #u Tyr Gly Ala Thr Phe            180       #           185       #           190Lys Asp Thr Ile Ile Leu Gly Met Val Ile Le #u Ala Val Val Pro Leu        195           #       200           #       205Leu Phe Leu Val Ile Pro Lys Met Lys Ile Se #r Ile Leu Asp Asp Pro    210               #   215               #   220Val Ala Ile Gly Leu Gly Leu His Val Gln Ar #g Met Lys Leu Ile Gln225                 2 #30                 2 #35                 2 #40Leu Ile Thr Ser Thr Ile Leu Val Ser Met Al #a Ile Ser Leu Val Gly                245   #               250   #               255Asn Ile Gly Phe Val Gly Leu Ile Ala Pro Hi #s Ile Ala Lys Thr Ile            260       #           265       #           270Val Arg Gly Ser Tyr Ala Lys Lys Leu Leu Me #t Ser Ala Met Ile Gly        275           #       280           #       285Ala Ile Ser Ile Val Ile Ala Asp Leu Ile Gl #y Arg Thr Leu Phe Leu    290               #   295               #   300Pro Lys Glu Val Pro Ala Gly Val Phe Ile Al #a Ala Phe Gly Ala Pro305                 3 #10                 3 #15                 3 #20Phe Phe Ile Tyr Leu Leu Leu Thr Val Lys Ly #s Leu                 325  #               330 <210> SEQ ID NO 21 <211> LENGTH: 1460<212> TYPE: DNA <213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 21taatgacact tattttttga aaataatagt aatatcattt tgttaaatga aa#gaataaag     60ctataataat tatagaataa ctatttaaag gagattataa acatgccaat ta#ttacagat    120gtttacgctc gcgaagtctt agactctcgt ggtaacccaa ctgttgaagt ag#aagtatta    180actgaaagtg gcgcatttgg tcgtgcatta gtaccatcag gtgcttcaac tg#gtgaacac    240gaagctgttg aattacgtga tggagacaaa tcacgttatt taggtaaagg tg#ttactaaa    300gcagttgaaa acgttaatga aatcatcgca ccagaaatta ttgaaggtga at#tttcagta    360ttagatcaag tatctattga taaaatgatg atcgcattag acggtactcc aa#acaaaggt    420aaattaggtg caaatgctat tttaggtgta tctatcgcag tagcacgtgc ag#cagctgac    480ttattaggtc aaccacttta caaatattta ggtggattta atggtaagca gt#taccagta    540ccaatgatga acatcgttaa tggtggttct cactcagatg ctccaattgc at#tccaagaa    600ttcatgattt tacctgtagg tgctacaacg ttcaaagaat cattacgttg gg#gtactgaa    660attttccaca acttaaaatc aattttaagc caacgtggtt tagaaactgc cg#taggtgac    720gaaggtggtt tcgctcctaa atttgaaggt actgaagatg ctgttgaaac aa#ttatccaa    780gcaatcgaag cagctggtta caaaccaggt gaagaagtat tcttaggatt tg#actgtgca    840tcatcagaat tctatgaaaa tggtgtatat gactacagta agttcgaagg cg#aacacggt    900gcaaaacgta cagctgcaga acaagttgac tacttagaac aattagtaga ca#aatatcct    960atcattacaa ttgaagacgg tatggacgaa aacgactggg atggttggaa ac#aacttaca   1020gaacgtatcg gtgaccgtgt acaattagta ggtgacgatt tattcgtaac aa#acactgaa   1080attttagcaa aaggtattga aaacggaatt ggtaactcaa tcttaattaa ag#ttaaccaa   1140atcggtacat taactgaaac atttgatgca atcgaaatgg ctcaaaaagc tg#gttacaca   1200gcagtagttt ctcaccgttc aggtgaaaca gaagatacaa caattgctga ta#ttgctgtt   1260gctacaaacg ctggtcaaat taaaactggt tcattatcac gtactgaccg ta#ttgctaaa   1320tacaatcaat tattacgtat cgaagatgaa ttatttgaaa ctgctaaata tg#acggtatc   1380aaatcattct ataacttaga taaataattt tctttataat caaatgctga ca#taatttta   1440 gttgaggatt attatgacgg             #                  #                 146 #0 <210> SEQ ID NO 22 <211> LENGTH: 434<212> TYPE: PRT <213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 22Met Pro Ile Ile Thr Asp Val Tyr Ala Arg Gl #u Val Leu Asp Ser Arg  1               5  #                 10  #                 15Gly Asn Pro Thr Val Glu Val Glu Val Leu Th #r Glu Ser Gly Ala Phe             20      #             25      #             30Gly Arg Ala Leu Val Pro Ser Gly Ala Ser Th #r Gly Glu His Glu Ala         35          #         40          #         45Val Glu Leu Arg Asp Gly Asp Lys Ser Arg Ty #r Leu Gly Lys Gly Val     50              #     55              #     60Thr Lys Ala Val Glu Asn Val Asn Glu Ile Il #e Ala Pro Glu Ile Ile 65                  # 70                  # 75                  # 80Glu Gly Glu Phe Ser Val Leu Asp Gln Val Se #r Ile Asp Lys Met Met                 85  #                 90  #                 95Ile Ala Leu Asp Gly Thr Pro Asn Lys Gly Ly #s Leu Gly Ala Asn Ala            100       #           105       #           110Ile Leu Gly Val Ser Ile Ala Val Ala Arg Al #a Ala Ala Asp Leu Leu        115           #       120           #       125Gly Gln Pro Leu Tyr Lys Tyr Leu Gly Gly Ph #e Asn Gly Lys Gln Leu    130               #   135               #   140Pro Val Pro Met Met Asn Ile Val Asn Gly Gl #y Ser His Ser Asp Ala145                 1 #50                 1 #55                 1 #60Pro Ile Ala Phe Gln Glu Phe Met Ile Leu Pr #o Val Gly Ala Thr Thr                165   #               170   #               175Phe Lys Glu Ser Leu Arg Trp Gly Thr Glu Il #e Phe His Asn Leu Lys            180       #           185       #           190Ser Ile Leu Ser Gln Arg Gly Leu Glu Thr Al #a Val Gly Asp Glu Gly        195           #       200           #       205Gly Phe Ala Pro Lys Phe Glu Gly Thr Glu As #p Ala Val Glu Thr Ile    210               #   215               #   220Ile Gln Ala Ile Glu Ala Ala Gly Tyr Lys Pr #o Gly Glu Glu Val Phe225                 2 #30                 2 #35                 2 #40Leu Gly Phe Asp Cys Ala Ser Ser Glu Phe Ty #r Glu Asn Gly Val Tyr                245   #               250   #               255Asp Tyr Ser Lys Phe Glu Gly Glu His Gly Al #a Lys Arg Thr Ala Ala            260       #           265       #           270Glu Gln Val Asp Tyr Leu Glu Gln Leu Val As #p Lys Tyr Pro Ile Ile        275           #       280           #       285Thr Ile Glu Asp Gly Met Asp Glu Asn Asp Tr #p Asp Gly Trp Lys Gln    290               #   295               #   300Leu Thr Glu Arg Ile Gly Asp Arg Val Gln Le #u Val Gly Asp Asp Leu305                 3 #10                 3 #15                 3 #20Phe Val Thr Asn Thr Glu Ile Leu Ala Lys Gl #y Ile Glu Asn Gly Ile                325   #               330   #               335Gly Asn Ser Ile Leu Ile Lys Val Asn Gln Il #e Gly Thr Leu Thr Glu            340       #           345       #           350Thr Phe Asp Ala Ile Glu Met Ala Gln Lys Al #a Gly Tyr Thr Ala Val        355           #       360           #       365Val Ser His Arg Ser Gly Glu Thr Glu Asp Th #r Thr Ile Ala Asp Ile    370               #   375               #   380Ala Val Ala Thr Asn Ala Gly Gln Ile Lys Th #r Gly Ser Leu Ser Arg385                 3 #90                 3 #95                 4 #00Thr Asp Arg Ile Ala Lys Tyr Asn Gln Leu Le #u Arg Ile Glu Asp Glu                405   #               410   #               415Leu Phe Glu Thr Ala Lys Tyr Asp Gly Ile Ly #s Ser Phe Tyr Asn Leu            420       #           425       #           430 Asp Lys

What is claimed is:
 1. An isolated polynucleotide encoding the aminoacid sequence of SEQ ID NO:12.
 2. The isolated polynucleotide of claim 1which is fused to a heterologous polynucleotide sequence.
 3. Theisolated polynucleotide of claim 2, wherein said heterologouspolynucleotide sequence encodes a heterologous polypeptide.
 4. Anisolated polynucleotide which is fully complementary to thepolynucleotide of claim
 1. 5. A method for making a recombinant vectorcomprising inserting the isolated polynucleotide of claim 1 into avector.
 6. A recombinant vector comprising the isolated polynucleotideof claim
 1. 7. The recombinant vector of claim 6, wherein said isolatedpolynucleotide is operably associated with a heterologous regulatorysequence that controls gene expression.
 8. An isolated recombinant hostcell comprising the isolated polynucleotide of claim
 1. 9. The isolatedrecombinant host cell of claim 8, wherein said polynucleotide isoperably associated with a heterologous regulatory sequence thatcontrols gene expression.
 10. A method for producing a polypeptide,comprising culturing the host cell of claim 8 under conditions suitableto produce the polypeptide encoded by said polynucleotide.
 11. Anisolated polynucleotide comprising the nucleotide sequence of SEQ IDNO:11.
 12. An isolated polynucleotide which is fully complementary tothe polynucleotide of claim
 11. 13. An isolated polynucleotideconsisting of the full length sequence of SEQ ID NO:11.
 14. An isolatedpolynucleotide consisting of a nucleic acid sequence encoding anepitope-bearing portion of the amino acid scquence of SEQ ID NO:12 isselected from the group consisting of: (a) Glu-7 to Asp-11; (b) IIe-18to Lys-20; (c) IIe-55 to Glu-59; (d) Ser-66 to Gly-77; (e) Thr-92 toThr-97; (f) Arg-123 to Asp-127; (g) Lys-154 to Asp-159; (h) IIe-166 toGln170; (i) Lys-618 to Thr-621; (j) Gly-628 to Val-632; (k) Asp-643 toLys-646; (l) Asp-667 to Arg-670; (m) Gly-681 to Asp-684; and (n) Asn-686to Lys-689.